Enzymes of the Tryptophan Synthetic Pathway in Pseudomonas putida

The first four enzymatic activities of the tryptophan synthetic pathway in Pseudomonas putida were found on separate molecules. Gel filtration and density gradient centrifugation experiments did not disclose any associations or aggregations among them. These findings contrast with the situation found in the enteric bacteria, where the first two activities are found in an aggregate and the third and fourth are catalyzed by a single enzyme. Tryptophan synthetase, the last enzyme of the pathway, consists of two dissociable components. The affinity of these components is less in P. putida than is the case in Escherichia coli.     

Evolutionary differences in chromosomal locations of four early genes of the tryptophan pathway in fluorescent pseudomonads: DNA sequences and characterization of Pseudomonas putida trpE and trpGDC.

Pseudomonas putida possesses seven structural genes for enzymes of the tryptophan pathway. All but one, trpG, which encodes the small (beta) subunit of anthranilate synthase, have been mapped on the circular chromosome. This report describes the cloning and sequencing of P. putida trpE, trpG, trpD, and trpC. In P. putida and Pseudomonas aeruginosa, DNA sequence analysis as well as growth and enzyme assays of insertionally inactivated strains indicated that trpG is the first gene in a three-gene operon that also contains trpD and trpC. In P. putida, trpE is 2.2 kilobases upstream from the trpGDC cluster, whereas in P. aeruginosa, they are separated by at least 25 kilobases (T. Shinomiya, S. Shiga, and M. Kageyama, Mol. Gen. Genet., 189:382-389, 1983). The DNA sequence in P. putida shows an open reading frame on the opposite strand between trpE and trpGDC; this putative gene was not characterized. Evidence is also presented for sequence similarities in the 5' untranslated regions of trpE and trpGDC in both pseudomonads; the function of these regions is unknown, but it is possible that they play some role in regulation of these genes, since all the genes respond to repression by tryptophan. The sequences of the anthranilate synthase genes in the fluorescent pseudomonads resemble those of p-aminobenzoate synthase genes of the enteric bacteria more closely than the anthranilate synthase genes of those organisms; however, no requirement for p-aminobenzoate was found in the Pseudomonas mutants created in this study.     

Activation of Chi recombinational hotspots by RecBCD-like enzymes from enteric bacteria

Chi sites, 5'G-C-T-G-G-T-G-G-3', enhance homologous recombination in Escherichia coli and are activated by the RecBCD enzyme. To test the ability of Chi to be activated by analogous enzymes from other bacteria, we cloned recBCD-like genes from diverse bacteria into an E. coli recBCD deletion mutant. Clones from seven species of enteric bacteria conferred to this deletion mutant recombination proficiency, Chi hotspot activity in lambda Red- Gam- vegetative crosses, and RecBCD enzyme activities, including Chi-dependent DNA strand cleavage. Three clones from Pseudomonas aeruginosa and Ps. putida conferred recombination proficiency and ATP-dependent nuclease activity, but neither Chi hotspot activity nor Chi-dependent DNA cleavage. These results imply that Chi has been conserved as a recombination-promoting signal for RecBCD-like enzymes in enteric bacteria but not in more distantly related bacteria such as Pseudomonas spp. We discuss the possibility that other, presently unknown, nucleotide sequences serve the same function as Chi in Pseudomonas spp.     

Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications.

Two anthranilate synthase gene pairs have been identified in Pseudomonas aeruginosa. They were cloned, sequenced, inactivated in vitro by insertion of an antibiotic resistance gene, and returned to P. aeruginosa, replacing the wild-type gene. One anthranilate synthase enzyme participates in tryptophan synthesis; its genes are designated trpE and trpG. The other anthranilate synthase enzyme, encoded by phnA and phnB, participates in the synthesis of pyocyanin, the characteristic phenazine pigment of the organism. trpE and trpG are independently transcribed; homologous genes have been cloned from Pseudomonas putida. The phenazine pathway genes phnA and phnB are cotranscribed. The cloned phnA phnB gene pair complements trpE and trpE(G) mutants of Escherichia coli. Homologous genes were not found in P. putida PPG1, a non-phenazine producer. Surprisingly, PhnA and PhnB are more closely related to E. coli TrpE and TrpG than to Pseudomonas TrpE and TrpG, whereas Pseudomonas TrpE and TrpG are more closely related to E. coli PabB and PabA than to E. coli TrpE and TrpG. We replaced the wild-type trpE on the P. aeruginosa chromosome with a mutant form having a considerable portion of its coding sequence deleted and replaced by a tetracycline resistance gene cassette. This resulted in tryptophan auxotrophy; however, spontaneous tryptophan-independent revertants appeared at a frequency of 10(-5) to 10(6). The anthranilate synthase of these revertants is not feedback inhibited by tryptophan, suggesting that it arises from PhnAB. phnA mutants retain a low level of pyocyanin production. Introduction of an inactivated trpE gene into a phnA mutant abolished residual pyocyanin production, suggesting that the trpE trpG gene products are capable of providing some anthranilate for pyocyanin synthesis.     

Expression of the argF gene of Pseudomonas aeruginosa in Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli

R' plasmids carrying argF genes from Pseudomonas aeruginosa strains PAO and PAC were transferred to Pseudomonas putida argF and Escherichia coli argF strains. Expression in P. putida was similar to that in P. aeruginosa and was repressed by exogenous arginine. Expression in E. coli was 2 to 4% of that in P. aeruginosa. Exogenous arginine had no effect, and there were no significant differences between argR' and argR strains of E. coli in this respect.     

Use of cloned genes of Pseudomonas TOL plasmid to effect biotransformation of benzoates to cis-dihydrodiols and catechols by Escherichia coli cells

DNA fragments containing the xylD and xylL genes, which specify the broad-specificity enzymes toluate-1,2-dioxygenase and 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid dehydrogenase, respectively, of TOL plasmid pWW0-161 of Pseudomonas putida have previously been cloned in the pBR322 vector plasmid (P.R. Lehrbach, J. Zeyer, W. Reinecke, H.-J. Knackmuss, and K. N. Timmis, J. Bacteriol. 158:1025-1032, 1984). In this study, Escherichia coli cells containing hybrid plasmids carrying the cloned xylD or xylDL genes quantitatively transformed 14C-ring- and 14C-carboxy-labeled benzoate to the pathway intermediates 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (cis-dihydrodiol) and catechol, respectively. Like P. putida cells, E. coli cells containing the xylD gene transformed a variety of chloro- and hydrocarbon-substituted benzoates. The toluate-1,2-dioxygenase produced in E. coli thus exhibited the broad-substrate-specificity properties of the enzyme in P. putida. Turnover rates by the enzymes in these two bacteria are compared.     

Expression of the lactose transposon Tn951 in Escherichia coli, Proteus and Pseudomonas

The control of beta-galactosidase specified by the lactose transposon Tn951 (inserted into RP1 to give pGC9114) has been studied in Escherichia coli K12, Proteus mirabilis, Pseudomonas aeruginosa and Pseudomonas putida; in the first two species comparison could be made with Flac. In E. coli K12, the Tn951 and chromosomally encoded enzymes showed marked qualitative differences in regulatio, the former giving a substantially lower maximum induced level and induction ratio. Several parameters were slightly affected by strain background. In P. mirabilis, beta-galactosidase control determined by both Flac (in accord with earlier work) and pGC9114 was markedly different from E. coli in that maximal induced levels were about an order of magnitude lower and the induction ratio was reduced to 3 to 5. In Ps. aeruginosa and Ps. putida, Tn951-specified lac expression was qualitatively similar to that in P. mirabilis. Possible reasons for anomalous expression in Proteus and Pseudomonas are discussed.     

Use of cloned genes of Pseudomonas TOL plasmid to effect biotransformation of benzoates to cis-dihydrodiols and catechols by Escherichia coli cells.

DNA fragments containing the xylD and xylL genes, which specify the broad-specificity enzymes toluate-1,2-dioxygenase and 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid dehydrogenase, respectively, of TOL plasmid pWW0-161 of Pseudomonas putida have previously been cloned in the pBR322 vector plasmid (P.R. Lehrbach, J. Zeyer, W. Reinecke, H.-J. Knackmuss, and K. N. Timmis, J. Bacteriol. 158:1025-1032, 1984). In this study, Escherichia coli cells containing hybrid plasmids carrying the cloned xylD or xylDL genes quantitatively transformed 14C-ring- and 14C-carboxy-labeled benzoate to the pathway intermediates 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (cis-dihydrodiol) and catechol, respectively. Like P. putida cells, E. coli cells containing the xylD gene transformed a variety of chloro- and hydrocarbon-substituted benzoates. The toluate-1,2-dioxygenase produced in E. coli thus exhibited the broad-substrate-specificity properties of the enzyme in P. putida. Turnover rates by the enzymes in these two bacteria are compared.     

Immunological and structural relatedness of catabolic ornithine carbamoyltransferases and the anabolic enzymes of enterobacteria.

Purified catabolic ornithine carbamoyltransferase of Pseudomonas putida and anabolic ornithine carbamoyltransferase (argF product) of Escherichia coli K-12 were used to prepare antisera. The two specific antisera gave heterologous cross-reactions of various intensities with bacterial catabolic ornithine carbamoyltransferases formed by Pseudomonas and representative organisms of other bacterial genera. The immunological cross-reactivity observed only between the catabolic ornithine carbamoyltransferases and the anabolic enzymes of enterobacteria suggests that these proteins share some structural similarities. Indeed, the amino acid composition of the anabolic ornithine carbamoyltransferase of E. coli K-12 (argF and argI products) closely resembles the amino acid compositions of the catabolic enzymes of Pseudomonas putida, Aeromonas formicans, Streptococcus faecalis, and Bacillus licheniformis. Comparison of the N-terminal amino acid sequence of the E. coli anabolic ornithine carbamoyltransferase with that of the A. formicans and Pseudomonas putida catabolic enzymes shows, respectively, 45 and 28% identity between the compared positions; the A. formicans sequence reveals 53% identity with the Pseudomonas putida sequence. These results favor the conclusion that anabolic ornithine carbamoyltransferases of enterobacteria and catabolic ornithine carbamoyltransferases derive from a common ancestral gene.     

Enzymatic production of L-tryptophan from DL-serine and indole by a coupled reaction of tryptophan synthase and amino acid racemase

Enzymatic production of L-tryptophan from DL-serine and indole by a coupled reaction of tryptophan synthase and amino acid racemase was studied. The tryptophan synthase (EC 4.2.1.20) of Escherichia coli catalyzed beta-substitution reaction of L-serine into L-tryptophan and the amino acid racemase (EC 5.1.1.10) of Pseudomonas putida catalyzed the racemization of D-serine simultaneously in one reactor. Under optimal conditions established for L-tryptophan production, a large-scale production of L-tryptophan was carried out in a 200-liter reactor using intact cells of E. coli and P. putida. After 24 h of incubation with intermittent indole feeding, 110 g liter-1 of L-tryptophan was formed in molar yields of 91 and 100% for added DL-serine and indole, respectively. Continuous production of L-tryptophan was also carried out using immobilized cells of E. coli and P. putida. The maximum concentration of L-tryptophan formed was 5.2 g liter-1 (99% molar yield for indole), and the concentration decreased to 4.2 g liter-1 after continuous operation for 20 days.     

Glutamine synthetase of pseudomonads: some biochemical and physicochemical properties.

The glutamine synthetases from several Pseudomonas species were purified to homogeneity, and their properties were compared with those reported for the enzymes from Escherichia coli and other gram-negative bacteria. The glutamine synthetase from Pseudomonas fluorescens was unique because it was nearly precipitated quantitatively as a homogeneous protein during dialysis of partially purified preparations against buffer containing 10 mM imidazole (pH 7.0) and 10 mM MnCl2. The glutamine synthetases from Pseudomonas putida and Pseudomonas aeruginosa were purified by affinity chromatography on Affi-blue gel. Dodecamerous forms of the E. coli and P. fluorescens glutamine synthetases had identical mobilities during polyacrylamide gel electrophoresis. Their dissociated subunits, however, migrated differently and were readily separated by electrophoresis on polyacrylamide gels containing 0.1% sodium dodecyl sulfate. This difference in subunit mobilities is not related to the state of adenylylation. Regulation of the Pseudomonas glutamine synthetase activity is mediated by an adenylylation-deadenylylation cyclic cascade system. A sensitive procedure was developed for measuring the average number of adenylylated subunits per enzyme molecule for the glutamine synthetase from P. fluorescens. This method takes advantage of the large differences in transferase activity of the adenylylated and unadenylylated subunits at pH 6.0 and of the fact that the activities of both kinds of subunits are the same at pH 8.45.     

Bioconversion of limonene to increased concentrations of perillic acid by Pseudomonas putida GS1 in a fed-batch reactor

Pseudomonas putida GS1 is able to convert limonene to perillic acid (up to 64 mM,(11 g/l) when the bacteria is cultivated in fed-batch culture with non-limiting amounts of glycerol. ammonium, and limonene. P. putida GS1 can use p-cymene as a single source of carbon and energy, and the enzymes that are responsible for the conversion of limonene to perillic acid belong to the degradation pathway of p-cymene. The p-cymene pathway of P putida GS1 is very similar, if not identical, to the cym pathway of P. putida F1. The latter strain, and a recombinant Escherichia coli strain that carried the genes of the cym pathway of P. putida Fl, also converted limonene to perillic acid. However, the final concentrations that were obtained in batch cultures with these two strains were lower than those obtained with P. putida GS1.     

Comparative Immunological Studies of Two Pseudomonas Enzymes

Crystalline preparations of muconate lactonizing enzyme and muconolactone isomerase, two inducible enzymes that catalyze successive steps in the catechol branch of the beta-ketoadipate pathway, were used to prepare antisera. Both enzymes were isolated from a strain of Pseudomonas putida biotype A. The antisera did not cross-react with enzymes of the same bacterial strain that catalyze the chemically analogous steps in the protocatechuate branch of the beta-ketoadipate pathway, carboxymuconate lactonizing enzyme and carboxymuconolactone decarboxylase. The antisera gave heterologous cross-reactions of varying intensities with the muconate lactonizing enzymes and muconolactone isomerases of P. putida biotype B, P. aeruginosa, P. stutzeri, and all biotypes of P. fluorescens, but did not cross-react with the isofunctional enzymes of P. acidovorans, of P. multivorans, and of two bacterial species that belong to other genera. The evolutionary and taxonomic implications of the findings are discussed.