Generation of a catR deficient mutant of P. putida KT2440 that produces cis, cis-muconate from benzoate at high rate and yield.

Pseudomonas putida KT2440-JD1 was derived from P. putida KT2440 after N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-mutagenesis and exposure to 3-fluorobenzoate (3-FB). The mutant was no longer able to grow using benzoate as a sole carbon source, but co-metabolized benzoate to cis, cis-muconate during growth on glucose, which accumulated in the growth medium. The specific production rate (q(pm)) was 0.18±0.03 g cis, cis-muconate/(g(DCW) h) in continuous cultures, and increased to 1.4 g cis, cis-muconate/(g(DCW) h) during wash-out cultivation. Transcriptome analysis showed that the cat operon was not induced in P. putida KT2440-JD1 in the presence of 5mM benzoate, due to a point mutation in the highly conserved DNA binding domain of the transcriptional regulator (catR) of the cat operon. The ben operon was highly expressed in the presence of benzoate in the mutant and its parental strain. This operon contains PP_3166 (catA2), which was shown to be a second catechol 1,2-dioxygenase besides catA. P. putida KT2440-JD1 is the first cis, cis-muconate-accumulating mutant that was characterized at the genetic level. The specific production rate achieved is at least eight times higher than those reported for other cis, cis-muconate-producing strains.

A limited LCA of bio-adipic acid: manufacturing the nylon-6,6 precursor adipic acid using the benzoic acid degradation pathway from different feedstocks.

A limited life cycle assessment (LCA) was performed on a combined biological and chemical process for the production of adipic acid, which was compared to the traditional petrochemical process. The LCA comprises the biological conversion of the aromatic feedstocks benzoic acid, impure aromatics, toluene, or phenol from lignin to cis, cis-muconic acid, which is subsequently converted to adipic acid through hydrogenation. Apart from the impact of usage of petrochemical and biomass-based feedstocks, the environmental impact of the final concentration of cis, cis-muconic acid in the fermentation broth was studied using 1.85% and 4.26% cis, cis-muconic acid. The LCA focused on the cumulative energy demand (CED), cumulative exergy demand (CExD), and the CO(2) equivalent (CO(2) eq) emission, with CO(2) and N(2) O measured separately. The highest calculated reduction potential of CED and CExD were achieved using phenol, which reduced the CED by 29% and 57% with 1.85% and 4.26% cis, cis-muconic acid, respectively. A decrease in the CO(2) eq emission was especially achieved when the N(2) O emission in the combined biological and chemical process was restricted. At 4.26% cis, cis-muconic acid, the different carbon backbone feedstocks contributed to an optimized reduction of CO(2) eq emissions ranging from 14.0 to 17.4 ton CO(2) eq/ton adipic acid. The bulk of the bioprocessing energy intensity is attributed to the hydrogenation reactor, which has a high environmental impact and a direct relationship with the product concentration in the broth.

Glycolytic pathway and hydrogen yield studies of the extreme thermophile Caldicellulosiruptor saccharolyticus.

NMR analysis of (13)C-labelling patterns showed that the Embden-Meyerhof (EM) pathway is the main route for glycolysis in the extreme thermophile Caldicellulosiruptor saccharolyticus. Glucose fermentation via the EM pathway to acetate results in a theoretical yield of 4 mol of hydrogen and 2 mol of acetate per mole of glucose. Previously, approximately 70% of the theoretical maximum hydrogen yield has been reached in batch fermentations. In this study, hydrogen and acetate yields have been determined at different dilution rates during continuous cultivation. The yields were dependent on the growth rate. The highest hydrogen yields of 82 to 90% of theoretical maximum (3.3 to 3.6 mol H(2) per mol glucose) were obtained at low growth rates when a relatively larger part of the consumed glucose is used for maintenance. The hydrogen productivity showed the opposite effect. Both the specific and the volumetric hydrogen production rates were highest at the higher growth rates, reaching values of respectively 30 mmol g(-1) h(-1) and 20 mmol l(-1) h(-1). An industrial process for biohydrogen production will require a bioreactor design, which enables an optimal mix of high productivity and high yield.

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.

Detoxification of reactive intermediates during microbial metabolism of halogenated compounds.

The reactivity and toxicity of metabolic intermediates that are generated by initial biotransformation reactions can be a major limiting factor for biodegradation of halogenated organic compounds. Recent work on the conversion of haloalkanes, chloroaromatics and chloroethenes indicates that microorganisms may become less sensitive to toxic effects either by using novel pathways that circumvent the generation of reactive intermediates or by producing modified enzymes that decrease the toxicity of such compounds.

Conversion of 3-chlorocatechol by various catechol 2,3-dioxygenases and sequence analysis of the chlorocatechol dioxygenase region of Pseudomonas putida GJ31.

Pseudomonas putida GJ31 contains an unusual catechol 2,3-dioxygenase that converts 3-chlorocatechol and 3-methylcatechol, which enables the organism to use both chloroaromatics and methylaromatics for growth. A 3.1-kb region of genomic DNA of strain GJ31 containing the gene for this chlorocatechol 2,3-dioxygenase (cbzE) was cloned and sequenced. The cbzE gene appeared to be plasmid localized and was found in a region that also harbors genes encoding a transposase, a ferredoxin that was homologous to XylT, an open reading frame with similarity to a protein of a meta-cleavage pathway with unknown function, and a 2-hydroxymuconic semialdehyde dehydrogenase. CbzE was most similar to catechol 2,3-dioxygenases of the 2.C subfamily of type 1 extradiol dioxygenases (L. D. Eltis and J. T. Bolin, J. Bacteriol. 178:5930-5937, 1996). The substrate range and turnover capacity with 3-chlorocatechol were determined for CbzE and four related catechol 2,3-dioxygenases. The results showed that CbzE was the only enzyme that could productively convert 3-chlorocatechol. Besides, CbzE was less susceptible to inactivation by methylated catechols. Hybrid enzymes that were made of CzbE and the catechol 2, 3-dioxygenase of P. putida UCC2 (TdnC) showed that the resistance of CbzE to suicide inactivation and its substrate specificity were mainly determined by the C-terminal region of the protein.

Symptoms of pervasive developmental disorders as observed in prediagnostic home videos of infants and toddlers.

OBJECTIVES: The objectives of this study were (1) to show prediagnostic abnormalities in social and communicative behaviors on home videos of children who later received a diagnosis of one of the pervasive developmental disorders (PDD) and (2) to demonstrate that prediagnostic abnormalities in social and communicative behaviors for children with PDD not otherwise specified will be less prominent than those in children with autistic disorder but still distinguishable from those of typically developing peers. STUDY DESIGN: Parents of children with PDD each submitted home videos of social events that were made when their child was between the ages of 12 and 30 months, before diagnosis. Two independent observers, unaware of the subjects' diagnoses or purpose of the study, scored the rates of specific anomalies in social and communicative behavior. Two additional observers scored the percentage of time the children were engaged socially or with objects. Data from the experimental group were compared with those of 25 age-matched children with no developmental disabilities. RESULTS: Significant differences were found between the rates of social engagement and 8 of the 25 specific behaviors of the children in whom PDD was later diagnosed and those of the typical children. The children later given the diagnosis of PDD not otherwise specified had mean frequencies of some social interactions and communicative skills that fell between those of children later given the diagnosis of autistic disorder and those of children with typical development. CONCLUSION: In our sample children in whom PDD was later diagnosed could be differentiated from their typically developing peers on the basis of specific anomalies noted in their social and communicative behaviors, especially joint attention. In our sample children with PDD not otherwise specified could have been further differentiated on the basis of the rates of social interaction. Careful assessment of social interaction and communicative behaviors may help to identify children with PDD before the age of 30 months.

Degradation of chloroaromatics: purification and characterization of a novel type of chlorocatechol 2,3-dioxygenase of Pseudomonas putida GJ31.

A purification procedure for a new kind of extradiol dioxygenase, termed chlorocatechol 2,3-dioxygenase, that converts 3-chlorocatechol productively was developed. Structural and kinetic properties of the enzyme, which is part of the degradative pathway used for growth of Pseudomonas putida GJ31 with chlorobenzene, were investigated. The enzyme has a subunit molecular mass of 33.4 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the native Mr value under nondenaturating conditions by gel filtration gave a molecular mass of 135 +/- 10 kDa, indicating a homotetrameric enzyme structure (4 x 33.4 kDa). The pI of the enzyme was estimated to be 7.1 +/- 0.1. The N-terminal amino acid sequence (43 residues) of the enzyme was determined and exhibits 70 to 42% identity with other extradiol dioxygenases. Fe(II) seems to be a cofactor of the enzyme, as it is for other catechol 2,3-dioxygenases. In contrast to other extradiol dioxygenases, the enzyme exhibited great sensitivity to temperatures above 40 degrees C. The reactivity of this enzyme toward various substituted catechols, especially 3-chlorocatechol, was different from that observed for other catechol 2,3-dioxygenases. Stoichiometric displacement of chloride occurred from 3-chlorocatechol, leading to the production of 2-hydroxymuconate.

Effect of trichloroethylene on the competitive behavior of toluene-degrading bacteria.

The influence of trichloroethylene (TCE) on a mixed culture of four different toluene-degrading bacterial strains (Pseudomonas putida mt-2, P. putida F1, P. putida GJ31, and Burkholderia cepacia G4) was studied with a fed-batch culture. The strains were competing for toluene, which was added at a very low rate (31 nmol mg of cells [dry weight] h). All four strains were maintained in the mixed culture at comparable numbers when TCE was absent. After the start of the addition of TCE, the viabilities of B. cepacia G4 and P. putida F1 and GJ31 decreased 50- to 1,000-fold in 1 month. These bacteria can degrade TCE, although at considerably different rates. P. putida mt-2, which did not degrade TCE, became the dominant organism. Kinetic analysis showed that the presence of TCE caused up to a ninefold reduction in the affinity for toluene of the three disappearing strains, indicating that inhibition of toluene degradation by TCE occurred. While P. putida mt-2 took over the culture, mutants of this strain which could no longer grow on p-xylene arose. Most of them had less or no meta-cleavage activity and were able to grow on toluene with a higher growth rate. The results indicate that cometabolic degradation of TCE has a negative effect on the maintenance and competitive behavior of toluene-utilizing organisms that transform TCE.

Microbial degradation of chloroaromatics: use of the meta-cleavage pathway for mineralization of chlorobenzene.

Pseudomonas putida GJ31 is able to simultaneously grow on toluene and chlorobenzene. When cultures of this strain were inhibited with 3-fluorocatechol while growing on toluene or chlorobenzene, 3-methylcatechol or 3-chlorocatechol, respectively, accumulated in the medium. To establish the catabolic routes for these catechols, activities of enzymes of the (modified) ortho- and meta-cleavage pathways were measured in crude extracts of cells of P. putida GJ31 grown on various aromatic substrates, including chlorobenzene. The enzymes of the modified ortho-cleavage pathway were never present, while the enzymes of the meta-cleavage pathway were detected in all cultures. This indicated that chloroaromatics and methylaromatics are both converted via the meta-cleavage pathway. Meta cleavage of 3-chlorocatechol usually leads to the formation of a reactive acylchloride, which inactivates the catechol 2,3-dioxygenase and blocks further degradation of catechols. However, partially purified catechol 2,3-dioxygenase of P. putida GJ31 converted 3-chlorocatechol to 2-hydroxy-cis,cis-muconic acid. Apparently, P. putida GJ31 has a meta-cleavage enzyme which is resistant to inactivation by the acylchloride, providing this strain with the exceptional ability to degrade both toluene and chlorobenzene via the meta-cleavage pathway.

Degradation of Toluene and Trichloroethylene by Burkholderia cepacia G4 in Growth-Limited Fed-Batch Culture.

Burkholderia (Pseudomonas) cepacia G4 was cultivated in a fed-batch bioreactor on either toluene or toluene plus trichloroethylene (TCE). The culture was allowed to reach a constant cell density under conditions in which the amount of toluene supplied equals the maintenance energy demand of the culture. Compared with toluene only, the presence of TCE at a toluene/TCE ratio of 2.3 caused a fourfold increase in the specific maintenance requirement for toluene from 22 to 94 nmol mg of cells (dry weight)(sup-1) h(sup-1). During a period of 3 weeks, approximately 65% of the incoming TCE was stably converted to unidentified products from which all three chlorine atoms were liberated. When toluene was subsequently omitted from the culture feed while TCE addition continued, mutants which were no longer able to grow on toluene or to degrade TCE appeared. These mutants were also unable to grow on phenol or m- or o-cresol but were still able to grow on catechol and benzoate. Plasmid analysis showed that the mutants had lost the plasmid involved in toluene monooxygenase formation (pTOM). Thus, although strain G4 is much less sensitive to TCE toxicity than methanotrophs, deleterious effects may still occur, namely, an increased maintenance energy demand in the presence of toluene and plasmid loss when no toluene is added.