Kelthane degradation by genetically engineered Pseudomonas aeruginosa BS827 in a soil ecosystem.

The aim of this study was to study the degradation of kelthane by Pseudomonas aeruginosa BS827, which carried the plasmid pBS3. This plasmid encodes naphthalene oxidation. The strain was able to survive in the presence of kelthane and to retain its degradative ability. Kelthane also stabilized the biodegradative plasmid that was preserved by 70 to 100% of the cell population. Cells deficient in Nah or Sal characters were less effective in degrading kelthane, whereas plasmid-free cells lost this ability completely. Evidently, the degradative activity of P. aeruginosa BS827 was conditioned by plasmid determinants coupled with genes of the plasmid pBS3 Nah region.     

Polymorphism in the interferon-alpha gene family.

A pronounced genetic polymorphism of the interferon type I gene family has been assumed on the basis of RFLP analysis of the genomic region as well as the large number of sequences published compared to the number of loci. However, IFNA2 is the only locus that has been carefully analyzed concerning gene frequency, and only naturally occurring rare alleles have been found. We have extended the studies on a variation of expressed sequences by studying the IFNA1, IFNA2, IFNA10, IFNA13, IFNA14, and IFNA17 genes. Genomic white-blood-cell DNA from a population sample of blood donors and from a family material were screened by single-nucleotide primer extension (allele-specific primer extension) of PCR fragments. Because of sequence similarities, in some cases "nested" PCR was used, and, when applicable, restriction analysis or control sequencing was performed. All individuals carried the interferon-alpha 1 and interferon-alpha 13 variants but not the LeIF D variant. At the IFNA2 and IFNA14 loci only one sequence variant was found, while in the IFNA10 and IFNA17 groups two alleles were detected in each group. The IFNA10 and IFNA17 alleles segregated in families and showed a close fit to the Hardy-Weinberg equilibrium. There was a significant linkage disequilibrium between IFNA10 and IFNA17 alleles. The fact that the extent of genetic polymorphism was lower than expected suggests that a majority of the previously described gene sequences represent nonpolymorphic rare mutants that may have arisen in tumor cell lines.     

Purification and characterization of 6-chlorohydroxyquinol 1,2-dioxygenase from Streptomyces rochei 303: comparison with an analogous enzyme from Azotobacter sp. strain GP1.

The enzyme which cleaves the benzene ring of 6-chlorohydroxyquinol was purified to apparent homogeneity from an extract of 2,4,6-trichlorophenol-grown cells of Streptomyces rochei 303. Like the analogous enzyme from Azotobacter sp. strain GP1, it exhibited a highly restricted substrate specificity and was able to cleave only 6-chlorohydroxyquinol and hydroxyquinol and not catechol, chlorinated catechols, or pyrogallol. No extradiol-cleaving activity was observed. In contrast to 6-chlorohydroxyquinol 1,2-dioxygenase from Azotobacter sp. strain GP1, the S. rochei enzyme had a distinct preference for 6-chlorohydroxyquinol over hydroxyquinol (kcat/Km = 1.2 and 0.57 s-1.microM-1, respectively). The enzyme from S. rochei appears to be a dimer of two identical 31-kDa subunits. It is a colored protein and was found to contain 1 mol of iron per mol of enzyme. The NH2-terminal amino acid sequences of 6-chlorohydroxyquinol 1,2-dioxygenase from S. rochei 303 and from Azotobacter sp. strain GP1 showed a high degree of similarity.     

Degradation of polychloroaromatic insecticides by Pseudomonas aeruginosa containing biodegradation plasmids

The work was aimed at studying the degradation of DDT, its metabolites and analogs by BS 816 and BS 827 strains constructed on the basis of Pseudomonas aeruginosa 640x strains and carrying biodegradation plasmids pBS2 and pBS3, respectively. DDT and kelthane were degraded by the BS 816 strain at a greater rate than by the parent culture. The investigation of enzymes involved in the oxidation of the aromatic cycle has shown that the plasmid-carrying strains possess the activity of metapyrocatechase and salicylate hydroxylase which is absent in P. aeruginosa 640x. The activity of pyrocatechase increased. In contrast to the parent strain where homogentisate induced only homogentisate oxygenase, this compound induces also metapyrocatechase in the constructed strains.     

Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus

The lignin-degrading, biopulping white-rot fungus Physisporinus rivulosus secretes several laccases of distinct features such as thermostability, extremely low pH optima and thermal activation for oxidation of phenolic substrates. Here we describe the cloning, heterologous expression and structural and enzymatic characterisation of two previously undescribed P. rivulosus laccases. The laccase cDNAs were expressed in the methylotrophic yeast Pichia pastoris either with the native or with Saccharomyces cerevisiae α-factor signal peptide. The specific activity of rLac1 and rLac2 was 5 and 0.3 μkat/μg, respectively. However, mutation of the last amino acid in the rLac2 increased the specific laccase activity by over 50-fold. The recombinant rLac1 and rLac2 enzymes demonstrated low pH optima with both 2,6-dimethoxyphenol (2,6-DMP) and 2,2′-azino-bis(3-ethylbenzathiazoline-6-sulfonate). Both recombinant laccases showed moderate thermotolerance and thermal activation at +60 °C was detected with rLac1. By homology modelling, it was deduced that Lac1 and Lac2 enzymes demonstrate structural similarity with the Trametes versicolor and Trametes trogii laccase crystal structures. Comparison of the protein architecture at the reducing substrate-binding pocket near the T1-Cu site indicated the presence of five amino acid substitutions in the structural models of Lac1 and Lac2. These data add up to our previous reports on laccase production by P. rivulosus during biopulping and growth on Norway spruce. Heterologous expression of the novel Lac1 and Lac2 isoenzymes in P. pastoris enables the detailed study of their properties and the evaluation of their potential as oxidative biocatalysts for conversion of wood lignin, lignin-like compounds and soil-polluting xenobiotics.     

Enzymes of a New Modified ortho-Pathway Utilizing 2-Chlorophenol in Rhodococcus opacus 1CP

Chlorocatechol 1,2-dioxygenase (CC 1,2-DO), chloromuconate cycloisomerase (CMCI), chloromuconolactone isomerase (CMLI), and dienolactone hydrolase (DELH), the key enzymes of a new modified ortho-pathway in Rhodococcus opacus 1CP cells utilizing 2-chlorophenol via a 3-chlorocatechol branch of a modified ortho-pathway, were isolated and characterized. CC 1,2-DO showed the maximum activity with 3-chlorocatechol; its activity with catechol and 4-chlorocatechol was 93 and 50%, respectively. The enzyme of the studied pathway had physicochemical properties intermediate between the pyrocatechase of ordinary and chlorocatechase of modified pathways described earlier for this strain. In contrast to the enzymes investigated earlier, CMCI of the new pathway exhibited high substrate specificity. The enzyme had K _m for 2-chloromuconate of 142.86 M, V _max = 71.43 U/mg, pH optimum around 6.0, and temperature optimum at 65°C. CMCI converted 2-chloromuconate into 5-chloromuconolactone. CMLI converted 5-chloromuconolactone into cis-dienolactone used as a substrate by DELH; this enzyme did not convert trans-dienolactone. DELH had Km for cis-dienolactone of 200 M, V _max = 167 U/mg, pH optimum of 8.6, and temperature optimum of 40°C. These results confirm the existence of a new modified ortho-pathway for utilization of 2-chlorophenol by R. opacus 1CP.     

19F NMR metabolomics for the elucidation of microbial degradation pathways of fluorophenols

Of all NMR-observable isotopes ¹⁹F is the one most convenient for studies on the biodegradation of environmental pollutants and especially for fast initial metabolic screening of newly isolated organisms. In the past decade we have identified the ¹⁹F NMR characteristics of many fluorinated intermediates in the microbial degradation of fluoroaromatics including especially fluorophenols. In the present paper we give an overview of results obtained for the initial steps in the aerobic microbial degradation of fluorophenols, i.e. the aromatic hydroxylation to di-, tri- or even tetrahydroxybenzenes ultimately suitable as substrates for the second step, ring cleavage by dioxygenases. In addition we present new results from studies on the identification of metabolites resulting from reaction steps following aromatic ring cleavage, i.e. resulting from the conversion of fluoromuconates by chloromuconate cycloisomerase. Together the presented data illustrate the potential of the ¹⁹F NMR technique for (1) fast initial screening of biodegradative pathways, i.e. for studies on metabolomics in newly isolated microorganisms, and (2) identification of relatively unstable pathway intermediates like fluoromuconolactones and fluoromaleylacetates. Journal of Industrial Microbiology & Biotechnology (2001) 26, 22–34. Received 20 April 2000/ Accepted in revised form 22 May 2000     

Heterogeneity of Rhodococcus opacus 1CP as a response to stress induced by chlorophenols

Dissociation of Rhodococcus opacus 1CP during culturing in different media (containing phenol and its monochlorinated derivatives as the sole source of carbon and energy) was studied. Three variants of strain 1CP (S1, S2, and R) differing in the morphology of cells and colonies, lipid composition, and manner of growth on phenol and monochlorophenols were isolated. It was shown that 2- and 4-chlorophenols were most actively degraded by the smooth (S) forms of the culture, and that the rough (R) form predominated when the culture was grown in a rich medium. The S forms differed from the R forms of the strain by an increased content of cardiolipin, fatty acids, and phosphatidylethanolamine.