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Pseudomonas putida uses L-lysine as the sole carbon and nitrogen source which preferentially requires its metabolism through two parallel pathways. In one of the pathways [delta]-aminovalerate is the key metabolite, whereas in the other L-lysine is racemized to D-lysine, and L-pipecolate and [alpha]-aminoadipate are the key metabolites. All the genes and enzymes involved in the D-lysine pathway, except for those involved in the conversion of D-lysine into [[DELTA].sup.1]-piperideine-2-carboxylate, have been identified previously (30). In this study we report that the conversion of D-lysine into [[DELTA].sup.1]-piperideine-2-carboxylate can be mediated by a D-lysine aminotransferase (PP3590) and a D-lysine dehydrogenase (PP3596). From a physiological point of view PP3596 plays a major role in the catabolism of D-lysine since its inactivation leads to a marked reduction in the growth rate with L- or D-lysine as the sole carbon and nitrogen source, whereas inactivation of PP3590 leads only to slowed growth. The gene encoding PP3590, called here amaC, forms an operon with dpkA, the gene encoding the enzyme involved in conversion of [[DELTA].sup.1]-piperideine-2-carboxylate to L-pipecolate in the D-lysine catabolic pathway. The gene encoding PP3596, called here amaD, is the fifth gene in an operon made up of seven open reading frames (ORFs) encoding PP3592 through PP3597. The dpkA amaC operon was transcribed divergently from the operon ORF3592 to ORF3597. Both promoters were mapped by primer extension analysis, which showed that the divergent -35 hexamers of these operon promoters were adjacent to each other. Transcription of both operons was induced in response to L- or D-lysine in the culture medium.

Cupriavidus necator(formerly Ralstonia eutropha) strain H850 is known to grow on biphenyl, and to co-oxidize congeners of polychlorinated biphenyls (PCBs). Using a Tn5-based minitransposon shuttle system and the TOL plasmid, the rational construction of hybrids of H850 was achieved by subsequent introduction of three distinct elements carrying 11 catabolic loci from three other biodegrading bacteria into the parent strain, finally yielding C. necator RW112. The new genetic elements introduced into H850 and its derivatives were tcbRCDEF, which encode the catabolic enzymes needed for chlorocatechol biodegradation under the control of a transcriptional regulator, followed by cbdABC, encoding a 2-halobenzoate dioxygenase, and xylXYZ, encoding a broad-spectrum toluate dioxygenase. The expression of the introduced genes was demonstrated by measuring the corresponding enzymic activities. The engineered strain RW112 gained the ability to grow on all isomeric monochlorobenzoates and 3,5-dichlorobenzoate, all monochlorobiphenyls, and 3,5-dichloro-, 2,3'-dichloro- and 2,4'-dichlorobiphenyl, without accumulation of chlorobenzoates. It also grew and utilized two commercial PCB formulations, Aroclor 1221 and Aroclor 1232, as sole carbon and energy sources for growth. This is the first report on the aerobic growth of a genetically improved bacterial strain at the expense of technical Aroclor mixtures.

The homogeneous preparation of type II hydride transferases of the old yellow enzyme family of flavoproteins, XenB of Pseudomonas putida, pentaerythritol tetranitrate reductase of Enterobacter cloacae, and N-ethylmaleimide reductase of Escherichia coli is reported. The flavoproteins responsible for the reduction of the polynitroaromatic compound 2,4,6-trinitro-toluene (TNT) have important environmental implications for TNT remediation.

A set of old yellow enzyme (OYE) homologs of Pseudomonas putida is utilized to study the potential complementary activities of multiple enzymes belonging to the same family within a single microorganism. The in vitro analysis of six OYE homologs identified in P. putida KT2440 offer insight into the catalytic activity of enzymes against 2,4,6-trinitrotoluene (TNT), which serves as a nitrogen source for microorganisms in vivo.

Unstable reduced derivatives of 2,4,6-trinitrotoluene (TNT) produced by microorganisms have been found to release nitrite by rearomatization and/or condensation. Here, we present further information regarding the novel mechanism of the condensation of reactive hydroxylaminodinitrotoluene and the Meisenheimer dihydride complex of TNT to produce two secondary diarylamine isomers. Using uniformly 15N-labeled (15N3) TNT, we show that the nitrite is being released by the condensation reaction and, also under environmental conditions, will originate from the microbiologically generated dihydride complex. [ABSTRACT FROM AUTHOR]

To investigate potential complementary activities of multiple enzymes belonging to the same family within a single microorganism, we chose a set of Old Yellow Enzyme (OYE) homologs of Pseudomonas putida. The physiological function of these enzymes is not well established; however, an activity associated with OYE family members from different microorganisms is their ability to reduce nitroaromatic compounds. Using an in silico approach, we identified six OYE homologs in P. putida KT2440. Each gene was subcloned into an expression vector, and each corresponding gene product was purified to homogeneity prior to in vitro analysis for its catalytic activity against 2,4,6-trinitrotoluene (TNT). One of the enzymes, called XenD, lacked in vitro activity, whereas the other five enzymes demonstrated type I hydride transferase activity and reduced the nitro groups of TNT to hydroxylaminodinitrotoluene derivatives. XenB has the additional ability to reduce the aromatic ring of TNT to produce Meisenheimer complexes, defined as type II hydride transferase activity. The condensations of the primary products of type I and type II hydride transferases react with each other to yield diarylamines and nitrite; the latter can be further reduced to ammonium and serves as a nitrogen source for microorganisms in vivo. [ABSTRACT FROM AUTHOR]

Polynitroaromatic explosives such as 2,4,6-trinitrophenol (picric acid) and 2,4,6-trinitrotoluene (TNT) are toxic and recalcitrant environmental pollutants. They persist in the environment due to the highly inactivated π system of their aromatic rings, which are inaccessible to dioxygenases that normally initiate the bacterial aerobic catabolism of (nitro-) aromatic compounds. Aside from reductive transformation of nitro side groups to hydroxylamines, trinitroarenes are prone to aromatic ring reductions by some flavin reductases to yield Meisenheimer mono and dihydride complexes. Here we show that the simultaneous accumulation of Meisenheimer complexes and aromatic hydroxylamines derived from TNT gives rise to the condensation of both types of reactive intermediates to secondary diarylamines and nitrite as the end-products of this environmentally relevant reaction sequence. As a consequence, overall mass balances of aerobic biotransformations of TNT become possible for the first time. In our study, the process of TNT activation was enzymatically initiated by the xenobiotic reductase B (XenB)-like flavin reductase of Pseudomonas putida JLR11 and then completed chemically by autodimerization. The structures of the formed end products were unequivocally elucidated by NMR. [ABSTRACT FROM AUTHOR]

The article discusses the growth of the engineered strain cupriavidus necator RW112 with chlorobenzoates and technical chlorobiphenyls. This study highlights the ability of the engineered strain RW112 to grow on all isomeric monochlorobenzoates, dichlorobenzoates and all monochlorobiphenyls without accumulation of chlorobenzoates. Engineered strain RW112 also grows and utilizes two commercial polychlorinated biphenyls formations that include Aroctor 1221 and Aroctor 1232.

Although much information on metabolic pathways within individual organisms is available, little is known about the pathways operating in natural communities in which extensive sharing of nutritional resources is the rule. In order to analyse such a consortium pathway, we have investigated the flow of 4chlorosalicylate as carbon substrate within a simple chemostat microbial community using [sup 13C-labelled metabolites and isotopic ratio mass spectrometric analysis of label enrichment in immunocaptured member populations of the community. A complex pathway network of carbon sharing was thereby revealed, involving two different metabolic routes, one of which is completely novel and involves the toxic metabolite protoanemonin. The high stability of the community results, at least in part, from interdependencies based on carbon sharing and the rapid removal of toxic metabolites. [ABSTRACT FROM AUTHOR]

Bacterial isolates obtained from polychlorophenol-contaminated sites in Finland (strain K101(T)) and from a Dutch drinking water well (strain A175(T)) were characterized taxonomically. 16S rRNA gene sequence analysis, determination of DNA G+C content, physiological characterization, estimation of the ubiquinone and polar lipid patterns and fatty acid content revealed that strains K101(T) and A175(T) were similar to Sphingomonas wittichii RW1(T) but also showed pronounced differences. The DNA G+C contents of the two novel strains were 63.6 and 66.1 mol%, respectively. On the basis of these results, two novel species of the genus Sphingomonas are described, for which the names Sphingomonas haloaromaticamans sp. nov. [type strain A175(T) (=DSM 13477(T)=CCUG 53463(T))] and Sphingomonas fennica sp. nov. [type strain K101(T) (=DSM 13665(T)=CCUG 53462(T))] are proposed.

A bacterial strain capable of degrading some monochlorinated dibenzofurans, designated RW16T, was isolated from aerobic River Elbe sediments. The strain was characterized based on 16S rRNA gene sequence analysis, DNA G+C content, physiological characteristics, polyamines, ubiquinone and polar lipid pattern and fatty acid composition. This analysis revealed that strain RW16T represents a novel species of the genus Sphingobium. The DNA G+C content of strain RW16T, 60.7 mol%, is the lowest yet reported for the genus. 16S rRNA gene sequence analysis placed strain RW16T as an outlier in the genus Sphingobium. The name Sphingobium aromaticiconvertens sp. nov. is proposed for this dibenzofuran-mineralizing organism, with type strain RW16T (=DSM 12677T=CIP 109198T).