The TOL plasmids of Pseudomonas putida encode enzymes required for the oxidation of toluene and other related aromatic compounds. These genes are organized into two operons, the xylUWCMABN operon (upper), and the xylXYZLTEGFJQKIH operon (lower). Here we report the nucleotide sequence of a 7107 bp segment of the TOL pDK1 plasmid encoding the region just upstream of the "upper" operon through the genes encoding xylUWCMA. Sequence analysis, comparison of base-usage patterns, codon-usage patterns, and intergenic distances between genes help support the idea that the "upper" and "lower" operons have evolved independently in different genetic backgrounds and have only more recently been brought together in TOL and related catabolic plasmids.

In der vorliegenden Arbeit wurden Untersuchungen zur mikrobiellen Aktivität von Pseudomonas putida DSM12735 durchgeführt. Als Messgröße diente die mikrobielle Wärmeleistung, basierend auf dem Stoffumsatz durch die Mikroorganismen. Ziel war es, die Vor- und Nachteile der verwendeten Kalorimeter herauszuarbeiten. Dafür wurden klassische Batch-Wachstumskurven aufgenommen. Ein weiteres Ziel bestand darin, eine Methode zur schnellen kalorimetrischen Detektion der mikrobiellen Aktivität insbesondere für die stationäre Phase zu entwickeln. In dieser Phase findet kein signifikanter Stoffumsatz statt. Durch das gezielte Auslösen einer zweiten Wachstumsphase und damit einem Stoffumsatz wird die mikrobielle Aktivität kalorimetrisch wieder messbar. Eingesetzt wurden folgende Kalorimeter: der Thermal Activity Monitor 2277 (TAM) mit den Kalorimetern Micro Reaction System 2250-4 ml und 2250-20 ml (kurz: TAM-4ml, TAM-20ml), das IC-Chip-Kalorimeter FCC22 (Institut für Physikalische Chemie, TU Freiberg) und das Kalorimeter Micro-DSC II (MDSC).

The nucleotide sequence for the pDKl TOL plasmid region encoding toluate-1,2-dioxygenase (Xy1XYZ, TO) was determined. TO is the first enzyme in the meta-cleavage operon, responsible for the conversion of toluates and benzoates to their carboxy-substituted diols. DNA sequence analysis revealed the presence of three open reading frames (ORF). The three ORFs correspond to xylX (1353 bp), xylY (486 bp) and xylZ (1008 bp), encoding predicted protein products of 51370 Da, 19368 Da and 36256 Da, respectively.

TOL plasmids encode enzymes responsible for utilization of toluene and related aromatic compounds by Pseudomonas putida, ultimately converting them to central metabolic intermediates. The nucleotide sequence for the 5.6 kb xylGFJQK region of the pDK1 TOL meta operon was determined. DNA sequence analysis revealed the presence of five open reading frames corresponding to xylG (1458 bp), xylF (846 bp), xylJ (783 bp), xylQ (936 bp) and xylK (1047 bp), encoding predicted protein products of 51.6, 31.3, 27.8, 32.8, and 36.6 kDa in size, respectively. The average G+C content of the xylLTEGFJQK region was 65.7%, somewhat higher than the 58.9% seen in the immediately upstream xylXYZ region and substantially more than the 50% G+C content reported for the upper TOL operon of this plasmid. Homology comparisons were made with genes and proteins of related catabolic plasmids. The dmpCDEFG and pWWO xylGFJQK regions exhibit consistently high levels of nucleotide and amino acid homology to pDK1 xylGFJQK throughout the entire region. In contrast, although the nucleotide sequence homology of the Acinetobacter atdCDE region to xylGFJ is high, the homology of atdFG to xylQK is markedly less. Such radical changes in homology between corresponding regions of different operons, combined with variable base and codon usage patterns within and between operons, provides additional support for the idea that the upper and lower operons encoding enzymes of aromatic pathways have evolved independently of one another and that these operons have continued to exchange genetic material with homologous expression units through a series of recombination events. Recombinant plasmids were constructed for individual expression of each of the xylGFJQK genes. HMSD (XylG) and HMSH (XylF) were partially purified and characterized with respect to substrate specificity and kinetic mechanism. Evidence was obtained suggesting that the HMSD reaction occurs via a steady state ordered mechanism or a random mechanism where binding of the first substrate effects the enzyme's affinity for the second substrate.

Like its enteric counterpart, aspartate transcarbamoylase (ATCase) from Pseudomonas putida is a dodecamer of two different polypeptides. Unlike the enterics, the Pseudomonas ATCase lacks regulatory polypeptides but employs instead inactive dihydroorotases for an active dodecamer. Previous work showed that PyrB contains not only the active site but also the effector binding sites for ATP, UTP and CTP at its N-terminus. In this work, 11 amino acids were deleted from the N-terminus of PyrB and the ATCase with the truncated protein was expressed in E. coli pyrB- and purified. The wild type enzyme was similarly treated. Velocity-substrate plots without effectors gave Michaelis-Menten kinetics in all cases. Deleting 11 amino acids did not affect dodecameric assembly but altered effector responses. When carbamoylphosphate was varied, the mutant enzyme was inhibited by UTP while the wild type enzyme was activated 2-fold. When the aspartate was varied, CTP had no effect on the mutant enzyme but strongly inhibited the wild type enzyme.

Cette étude se situe au carrefour d'une problématique opérationnelle de gestion des sols de bassins d'infiltration d'eaux pluviales et d'une thématique plus théorique sur le transport colloïdal. En effet, malgré la mise en évidence du rôle parfois prédominant des colloïdes dans le transport de polluants, les mécanismes considérés prennent encore peu en compte cette forme de transport et les conditions sous lesquelles il prédomine restent mal définies. Dans ce cadre, l'objectif principal de ces travaux est la caractérisation du transport colloïdal d'un métal caractéristique des pollutions d'eaux pluviales urbaines, le zinc. Les colloïdes considérés sont des bactéries de l'espèce Pseudomonas putida qui sont présentes dans le milieu poreux sous forme de biofilm. Les essais ont été réalisés en colonnes de sable de Loire, non saturées en eau. Des expériences complémentaires en réacteurs fermés ont permis de mieux caractériser la réactivité des surfaces mises en jeu. Les résultats permettent d'apporter des éléments de réponse à trois grandes questions qui sont en fait les conditions nécessaires pour qu'il y ait transport colloïdal : réactivité des colloïdes pour les polluants, mouvement des colloïdes, et transport associé. Tout d'abord, les titrations de surface et les essais d'adsorption ont montré la grande réactivité des surfaces en particulier du biofilm. Ensuite la composition de la solution en contact avec le milieux poreux et sa force ionique influencent largement la stabilité du biofilm et donc la formation de colloïdes mobiles. Ainsi les conditions sont réunies pour observer le transport associé du zinc et des bactéries; celui-ci n'a pas été directement quantifié, mais observé par des méthodes indirectes. Il apparaît donc nécessaire aujourd'hui de tenir compte, sous certaines conditions, du transport facilité par les colloïdes bactériens dans les sols de bassins d'infiltration.

The shikimate pathway links carbohydrate metabolism to biosynthesis of the aromatic amino acids in plants, fungi, bacteria and apicomplexan parasites. The pathway has seven enzymatic steps which convert erythrose-4-phosphate and phosphoenolpyruvate to chorismate, the precursor of tyrosine, tryptophan and phenylalanine. Due to the absence of the pathway in mammalian species, the enzymes are attractive targets for herbicides and antimicrobials. Shikimate dehydrogenase (SDH) catalyses the fourth step, the NADP-dependent reversible reduction of 3-dehydroshikimate to shikimate. Five SDH homologs – AroE, Ael1, YdiB, RifI and SdhL – have been identified through kinetic analysis and phylogenetic studies in the bacterium Pseudomonas putida. SDH homolog gene knockouts (KO) were used to characterize their functions. The AroE KO and Ael1 KO were successfully constructed via gene SOEing of the SDH homolog with a gentamycin antibiotic cassette and homologous recombination via electroporation into WT P. putida KT2440. Preliminary characterization tested KO growth, auxotroph recovery and fluorescent activity.

The shikimate pathway links carbohydrate metabolism to biosynthesis of the aromatic amino acids in plants, fungi, bacteria and apicomplexan parasites. The pathway has seven enzymatic steps which convert erythrose-4-phosphate and phosphoenolpyruvate to chorismate, the precursor of tyrosine, tryptophan and phenylalanine. Due to the absence of the pathway in mammalian species, the enzymes are attractive targets for herbicides and antimicrobials. Shikimate dehydrogenase (SDH) catalyses the fourth step, the NADP-dependent reversible reduction of 3-dehydroshikimate to shikimate. Five SDH homologs – AroE, Ael1, YdiB, RifI and SdhL – have been identified through kinetic analysis and phylogenetic studies in the bacterium Pseudomonas putida. SDH homolog gene knockouts (KO) were used to characterize their functions. The AroE KO and Ael1 KO were successfully constructed via gene SOEing of the SDH homolog with a gentamycin antibiotic cassette and homologous recombination via electroporation into WT P. putida KT2440. Preliminary characterization tested KO growth, auxotroph recovery and fluorescent activity.

Stuttgart, Univ., Diss., 2002.

Thesis (Ph. D)--Rijksuniversiteit te Utrecht, 1983. / Summary in Dutch. Stellingen inserted. Includes bibliographical references.