Degradation of the herbicide bromoxynil inPseudomonas putida

Biological conversion of the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) was studied in a batch culture ofPseudomonas putida by using HPLC. The process had a cometabolic character and proceeded only in the presence of another, simultaneously metabolizable, carbon and energy source. The intensity of degradation correlated with the growth rate, the degradation stopping when the cosubstrate becomes exhausted or the pH value of the medium falls below 6.5. In a medium with glucose, no lag phase longer than one day was observed concerning growth, sugar and herbicide consumption and formation of metabolic herbicide derivatives (3,5-dibromo-4-hydroxybenzamide and 3,5-dibromo-4-hydroxybenzoic acid). In a medium with ribose, the initial lag of the above processes took 2 d. No formation of other degradation products was detected. Growth inhibition was proportional to the concentration of bromoxynil. Translated by Č. Novotny     

Maintenance and killing efficiency of conditional lethal constructs inPseudomonas putida

Summary Conditional lethal (suicidal) genetic constructs were designed and employed in strains of Pseudomonads as models for containment of geneticallyengineerd microbes that may be deliberately released into the environment. A strain ofPseudomonas putida was formed with a suicide vector designated pBAP24h that was constructed by cloning the host killing gene (hok) into the RSF1010 plasmid pVDtac24 and placing it under the control of thetac promoter. Afterhok induction inP. putida only 40% of surviving cells continued to bear thehok sequences within 4 h of induction; in contrast, 100% of the cells in uninduced controls borehok. A few survivors that demonstrated resistance tohok-induced killing developed inP. putida, which may have been due to a mutation or physiological adaptation that rendered the membrane resistant tohok. Conditional lethal strains ofP. putida also were formed by insertinggef (a chromosomal homolog ofhok) under the control of thetac promoter into the chromosome using a transposon. Constructs with chromosomalgef, as well as an RK2-derived plasmid construct containinggef, were only marginally more stable than thehok constructs; they were effective in killingP. putida when induced and within 2 h post-induction killing from eithergef construct resulted in a 10³–10⁵-fold reduction in viable cell count compared to uninduced controls.     

Regulation of urea uptake inPseudomonas aeruginosa

The energy-dependent urea permease was studied in two strains ofPseudomonas aeruginosa, measuring the uptake (transport and metabolism) of¹⁴C-urea. In both strains urea uptakein vivo and urease activityin vitro differed significantly with respect to kinetic parameters, temperature and pH dependence and response to metabolic inhibitors. Ammonium strongly interfered both with the expression of the urea uptake system and its activity. The inhibition of the uptake activity by ammonium was partially relieved by hydraziniumsulfate, which prevented the translocation of ammonium into the cell, and in a methylammonium/ammonium transport-defective mutant of strain DSM 50071. Furthermore, methionine-sulfoximine, which prevented the intracellular glutamine formation from ammoniumvia inhibition of glutamine synthetase, relieved the inhibition of urea uptake by ammonium. These findings suggested that urea uptake activity inP. aeruginosa is regulated by intracellular glutamine.Abbreviations CCCP carbonylcyanide-m-chlorphenylhydrazone - DCCD dicyclohexylcarbodiimide - GS glutamine synthetase - MSX methionine-sulfoximine     

Effect of dissolved oxygen concentration on the metabolism of glucose inPseudomonas putida BM014

The effect of dissolved oxygen concentration on the metabolism of glucose inPseudomonas putida BM014 was investigated. Glucose was completely converted to 2-ketogluconatevia extracellular oxdative pathway and then taken up for cell growth under the condition of sufficient dissolved oxygen concentration. On the other hand, oxygen limitation below dissolved oxygen tension (DOT) value of 20% of air saturation caused the shift of glucose metabolism from the extracellular oxidative pathway to the intracellular phosphorylative pathway. Specific activities of hexokinase and gluconate kinase in intracellular phosphorylation pathway decreased as the DOT increased, while 2-ketogluconokinase activity in extracellular oxidative pathway increased under the same condition. This result can be usefully applied to microbial transformation of glucose to 2-ketogluconate, the synthetic precursor for iso-vitamine C, with almost 100% yieldvia extracellular oxidation by simple DOT control.     

Multiple outer membrane receptors for uptake of ferric pseudobactins inPseudomonas putida WCS358

Under iron limitationPseudomonas putida WCS358 produces a fluorescent siderophore, pseudobactin 358, which, after complexing iron, is transported back into the cell via the specific outer membrane receptor PupA. In addition, this strain has the capacity to take up iron via a large variety of siderophores produced by other fluorescent pseudomonads. Putative receptor genes for such siderophores were identified in the chromosome of strain WCS358 by PCR using primers matching two domains conserved in four ferric pseudobactin receptors, including PupA. Eleven amplification products within the expected size range were obtained. Sequence analysis confirmed that the products were derived from genes encoding outer membrane receptors. Two complete receptor genes were isolated from a genomic library ofP. putida WCS358. Both protein products are involved in the transport of a limited number of specific ferric pseudobactins. These results indicate that the ability ofP. putida WCS358 to exploit many different heterologous pseudobactins is related to the presence of multiple outer membrane receptor proteins.     

Hyperinduction of enzymes of the phosphorylative pathway of glucose dissimilation inPseudomonas cepacia

Glucose-dehydrogenase-deficient (Gcd^–) strains ofPseudomonas cepacia 249 compensated for loss of operation of the direct oxidative pathway by expanding the phosphorylative pathway. When grown on glucose, they had between two- and fourfold higher than normal levels of glucokinase and NAD-linked glucose-6-phosphate dehydrogenase activity and a comparable increase in capacity to transport glucose. Similar expansion of the phosphorylative pathway was noted when the wild type was grown on cellobiose or trehalose. Gcd^– strains grew normally on cellobiose and trehalose, but not if also deficient in glucokinase; this indicates that the disaccharides were converted to glucose and metabolized via the phosphorylative pathway. The expansion of the phosphorylative pathway during growth of the wild type on disaccharides or of Gcd^– mutants on glucose was a consequence of hyperinduction of pathway enzymes. Other compounds that promoted such hyperinduction included aromatic conjugates of glucose such as arbutin and salicin, and mannose. Under conditions leading to expansion of the phosphorylative pathway, enzymes related to the direct oxidative pathway, such as gluconate dehydrogenase and the 6-phosphogluconate dehydrogenase active with NAD, were not formed. The results indicate that intracellular glucose and extracellular glucose are metabolized to 6-phosphogluconate via different routes.     

Regulation of enzymes of the 3,5-xylenol-degradative pathway in Pseudomonas putida: evidence for a plasmid.

Constitutive synthesis of enzymes responsible for methyl group oxidation in 3,5-xylenol degradation and an associated p-cresol methylhydroxylase in Pseudomonas putida NCIB 9869 was shown by their retention at high specific activities in cells transferred from 3,5-xylenol medium to glutamate medium. The specific activities of other enzymes of the 3,5-xylenol pathway declined upon removal of aromatic substrate, consistent with their inducible control. Specific activities of the methyl-oxidizing enzymes showed an eventual decline concomitant with a decrease in the fraction of bacteria capable of growth with 3,5-xylenol; a simultaneous loss of the ability to grow with m-hydroxybenzoate was also observed. The property of 3,5-xylenol utilization could be transferred to another strain of P. putida. It is proposed that enzymes of the 3,5-xylenol pathway and those for conversion of p-cresol to p-hydroxybenzoate are plasmid encoded, that the early methyl-oxidizing enzymes are expressed constitutively, and that the later enzymes are inducible.     

Enhancement ofcis,cis-muconate productivity by overexpression of catechol 1,2-dioxygenase inPseudomonas putida BCM114

For enhancement ofcis,cis-muconate productivity from benzoate, catechol 1,2-dioxygenase (C12O) which catalyzes the rate-limiting step (catechol conversion tocis,cis-muconate) was cloned and expressed in recombinantPseudomonas putida BCM114. At higher benzoate concentrations (more than 15 mM),cis,cis-muconate productivity gradually decreased and unconverted catechol was accumulated up to 10 mM in the case of wildtypeP. putida BM014, whereascis,cis-muconate productivity continuously increased and catechol was completely transformed tocis,cis-muconate forP. putida BCM114. Specific C12O activity ofP. putida BCM114 was about three times higher than that ofP. putida BM014, and productivity was enhanced more than two times.     

Organization and expression inPseudomonas putida of the gene cluster involved in cephalosporin biosynthesis fromLysobacter lactamgenus YK90

TheLysobacter lactamgenus YK90pcbAB gene encoding -(l--aminoadipyl)-l-cysteinyl-d-valine (ACV) synthetase is located immediately upstream of thepcbC gene in the same orientation in the gene cluster involved in cephalosporin biosynthesis. ThepcbAB gene encodes a large polypeptide composed of 3722 amino acid residues with a molecular mass of 411 593 Da. The predicted amino acid sequence has a high degree of similarity with those of known ACV synthetases from fungi and actinomycetes. Within thepcbAB amino acid sequence, three conserved and repeated domains of about 600 amino acids were identified. The domains also share a high degree of similarity with non-ribosomal peptide synthetases such as gramicidin synthatase 2 ofBacillus brevis. ThepcbAB gene was expressed under the control of thelac promoter inPseudomonas putida. Expression of the gene cluster involved in cephalosporin biosynthesis inP. putida led to the accumulation of -lactam antibiotics. Deletion analysis of an open-reading frame located between thecefE andcefD genes from the gene cluster revealed that it encoded deacetylcephalosporin C synthetase (cefF). From the results presented here and those of previous studies, the genes involved in cephalosporin biosynthesis inL. lactamgenus appear to be clustered in the orderpcb AB-pcbC- cefE-cefF-cefD-bla in the same orientation within a 17-kb region of DNA.     

Regulation of the degradative pathway enzymes coded for by the TOL plasmid (pWWO) from Pseudomonas putida mt-2

Pseudomonas putida mt-2 carries a plasmid (TOL, pWWO) which codes for a single set of enzymes responsible for the catabolism of toluene and m- and p-xylene to central metabolites by way of benzoate and m- and p-toluate, respectively, and subsequently by a meta cleavage pathway. Characterization of strains with mutations in structural genes of this pathway demonstrates that the inducers of the enzymes responsible for further degradation of m-toluate include m-xylene, m-methylbenzyl alcohol, and m-toluate, whereas the inducers of the enzymes responsible for oxidation of m-xylene to m-toluate include m-xylene and m-methylbenzyl alcohol but not m-toluate. A regulatory mutant is described in which m-xylene and m-methylbenzyl alcohol no longer induce any of the pathway enzymes, but m-toluate is still able to induce the enzymes responsible for its own degradation. Among revertants of this mutant are some strains in which all the enzymes are expressed constitutively and are not further induced by m-xylene. A model is proposed for the regulation of the pathway in which the enzymes are in two regulatory blocks, which are under the control of two regulator gene products. The model is essentially the same as proposed earlier for the regulation of the isofunctional pathway on the TOL20 plasmid from P. putida MT20.     

Regulation of leucine catabolism in Pseudomonas putida

The generation time of Pseudomonas putida with l-leucine was 20 h in synthetic media but only 3 h with d-leucine. Slow growth in the presence of l-leucine was partially overcome by addition of 0.1 mM amounts of either d-valine, l-valine, or 2-ketoisovalerate. The activities of five enzymes which take part in the oxidation of leucine by P. putida were measured under various conditions of growth. Four enzymes were induced by growth with dl-leucine as sole source of carbon: d-amino acid dehydrogenase, branched-chain keto acid dehydrogenase, 3-methylcrotonyl-coenzyme A carboxylase, and 3-hydroxy-3-methylglutaryl-coenzyme A lyase. The segment of the pathway required for oxidation of 3-methylcrotonate was induced by growth on isovalerate or 3-methylcrotonate without formation of the preceding enzymes. The synthesis of carboxylase and lyase appeared to have been repressed by the addition of l-glutamate or glucose to cells growing on dl-leucine as the sole carbon source. Mutants unable to grow at the expense of isovalerate had reduced levels of carboxylase and lyase, whereas the levels of three enzymes common to the catabolism of all three branched-chain amino acids and those of two isoleucine catabolic enzymes were normal.     

Regulation of valine catabolism in Pseudomonas putida

The activities of six enzymes which take part in the oxidation of valine by Pseudomonas putida were measured under various conditions of growth. The formation of four of the six enzymes was induced by growth on d- or l-valine: d-amino acid dehydrogenase, branched-chain keto acid dehydrogenase, 3-hydroxyisobutyrate dehydrogenase, and methylmalonate semialdehyde dehydrogenase. Branched-chain amino acid transaminase and isobutyryl-CoA dehydrogenase were synthesized constitutively. d-Amino acid dehydrogenase and branched-chain keto acid dehydrogenase were induced during growth on valine, leucine, and isoleucine, and these enzymes were assumed to be common to the metabolism of all three branched-chain amino acids. The segment of the pathway required for oxidation of isobutyrate was induced by growth on isobutyrate or 3-hydroxyisobutyrate without formation of the preceding enzymes. d-Amino acid dehydrogenase was induced by growth on l-alanine without formation of other enzymes required for the catabolism of valine. d-Valine was a more effective inducer of d-amino acid dehydrogenase than was l-valine. Therefore, the valine catabolic pathway was induced in three separate segments: (i) d-amino acid dehydrogenase, (ii) branched-chain keto acid dehydrogenase, and (iii) 3-hydroxyisobutyrate dehydrogenase plus methylmalonate semialdehyde dehydrogenase. In a study of the kinetics of formation of the inducible enzymes, it was found that 3-hydroxyisobutyrate and methylmalonate semialdehyde dehydrogenases were coordinately induced. Induction of enzymes of the valine catabolic pathway was studied in a mutant that had lost the ability to grow on all three branched-chain amino acids. Strain PpM2106 had lowered levels of branched-chain amino acid transaminase and completely lacked branched-chain keto acid dehydrogenase when grown in medium which contained valine. Addition of 2-ketoisovalerate, 2-ketoisocaproate, or 2-keto-3-methylvalerate to the growth medium of strain PpM2106 resulted in induction of normal levels of branched-chain keto acid dehydrogenase; therefore, the branched-chain keto acids were the actual inducers of branched-chain keto acid dehydrogenase.     

Stereospecific enzymes in the degradation of aromatic compounds by pseudomonas putida

Two reactions in the catabolism of catechol by meta-fission, namely, hydration of 2-oxopent-4-enoate (vinylpyruvate) and aldol fission of the product, are catalyzed by stereospecific enzymes. The absolute configuration of this hydration product was shown to be l(S)-4-hydroxy-2-oxopentanoate. Vinylpyruvate hydratase, purified almost to homogeneity, had a molecular weight of about 287,000 and was dissociated in sodium dodecyl sulfate, without prior treatment with mercaptoethanol, into a species with an approximate molecular weight of 28,000. The hydratase was highly specific for its substrates; thus, although 2-oxo-cis-hex-4-enoate was also hydrated, structurally similar compounds such as the trans isomer, vinylacetic and crotonic acids, and the ring-fission products of catechol and methylcatechols were not attacked. Vinylpyruvate hydratase was activated by Mn(2+) ions. On the basis of these observations, a mechanism is proposed which closely resembles that for 4-hydroxy-2-oxopentanoate aldolase. A possible evolutionary connection between functionally related, divalent cation-activated hydro-lyases and aldolases is discussed. It was also demonstrated that l-(S)-4-hydroxy-2-oxohexanoate is the biologically active enantiomer of this hydroxy acid.     

The involvement of an enantioselective transaminase in the metabolism of D-3- and D-4-hydroxyphenylglycine inPseudomonas putida LW-4

Summary Pseudomonas putida LW-4, isolated on D-phenylglycine as sole carbon and energy source, was also able to grow on D-3-and D-4-hydroxyphenylglycine. Both D-3-and D-4-hydroxyphenylglycine were initially converted to the corresponding hydroxyphenylglyoxylates by means of an enantioselective transaminase. Subsequently, the hydroxyphenylglyoxylates were decarboxylated and then oxidized to 3-and 4-hydroxybenzoate, respectively. These latter compounds in turn were oxidized by NADPH-dependent hydroxylases to protocatechuate, which was further oxidized via an intradiol cleavage. Preliminary experiments with cell extracts in which the 4-hydroxyphenylglyoxylate decarboxylase was partially removed by an ammonium sulfate fractionation showed that D-4-hydroxyphenylglyoxycine could be formed from 4-hydroxyphenylglyoxylate by the enantioselective transaminase.     

Ni2+-uptake inPseudomonas putida strain S4: a possible role of Mg2+-uptake pump

Essential metal ion homeostasis is based on regulated uptake of metal ions, both during its scarcity and abundance.Pseudomonas putida strain S4, a multimetal resistant bacterium, was employed to investigate Ni²⁺ entry into cells. It was observed that Mg²⁺ regulates the entry of Ni²⁺ and by this plays a protective role to minimize Ni²⁺ toxicity in this strain. This protection was evident in both growth as well as viability. Intracellular accumulation of Ni²⁺ varied in accordance with Mg²⁺ concentrations in the medium. It was hypothesized that Ni²⁺ enters the cell using a broad Mg²⁺ pump, i.e. the CorA system, as the CorA inhibitor, i.e. Co(III) Hex, also inhibits Ni²⁺ uptake. This led to the inference that Mg²⁺-based protection was basically due to competitive inhibition of Ni²⁺ uptake. We also show that Zn²⁺ can further regulate the entry of Ni²⁺     

Regulation of alkane oxidation in Pseudomonas putida.

We have studied the appearance of whole-cell oxidizing activity for n-alkanes and their oxidation products in strains of Pseudomonas putida carrying the OCT plasmid. Our results indicate that the OCT plasmid codes for inducible alkane-hydroxylating and primary alcohol-dehydrogenating activities and that the chromosome codes for constitutive oxidizing activities for primary alcohols, aliphatic aldehydes, and fatty acids. Mutant isolation confirms the presence of an alcohol dehydrogenase locus on the OCT plasmid and indicated the presence of multiple alcohol and aldehyde dehydrogenase loci on the P. putida chromosome. Induction tests with various compounds indicate that inducer recognition has specificity for chain length and can be affected by the degree of oxidation of the carbon chain. Some inducers are neither growth nor respiration substrates. Growth tests with and without a gratuitous inducer indicate that undecane is not a growth substrate because it does not induce alkane hydroxylase activity. Using a growth test for determining induction of the plasmid alcohol dehydrogenase it is possible to show that heptane induces this activity in hydroxylase-negative mutants. This suggests that unoxidized alkane molecules are the physiological inducers of both plasmid activities.     

Regulation of food enzymes

In this review the development of federal policy for the regulation of enzymes is summarized. Defining enzymes as food additives and considerations of the health and safety aspects of employing enzymes in food processing are described. A review of the status of GRAS petitions dealing with enzymes by the governmental review branch is discussed, along with recent developments recommending major changes in the Federal Food, Drug and Cosmetics Act, which is the basic law covering food safety.