1,2-Dihydroxynaphthalene dioxygenase was purified to homogeneity from a bacterium that degrades naphthalenesulfonic acids (strain BN6). The enzyme requires Fe2" for maximal activity and consists of eight identical subunits with a molecular weight of about 33,000. Analysis of the NH2-terminal amino acid sequence revealed a high degree of homology (22 of 29 amino acids) with the NH2-terminal amino acid sequence of 2,3-dihydroxybiphenyl dioxygenase from strain Pseudomonas paucimobiis Ql. 1,2-Dihydroxynaphthalene dioxygenase from strain BN6 shows a wide substrate specificity and also cleaves 5-, 6-, and 7-hydroxy-1,2-dihydroxynaphthalene, 2,3-and 3,4-dihydroxybiphenyl, catechol, and 3-methyl-and 4-methylcatechol. Similar activities against the hydroxy-1,2-dihydroxynaphthalenes were also found in cell extracts from naphthalenedegrading bacteria.Amino-and hydroxynaphthalenesulfonic acids (ANS and HNS, respectively) serve as building blocks for the largescale synthesis of azo dyes. Since arylsulfonates are very rare among natural compounds (21), naphthalenesulfonic acids are referred to as xenobiotics. Arylsulfonates are major pollutants of the environment, and the contamination of the Rhine River by sulfur-organic compounds is largely due to this class of compounds (25). Nevertheless, bacteria which degrade naphthalenesulfonic acids have repeatedly been isolated (4, 5, 29-31, 41, 43). Brilon et al. (4, 5) selected pseudomonads which degraded naphthalene-2-sulfonic acid (2NS) and naphthalene-1-sulfonic acid (1NS). The initial reaction in the degradation of iNS and 2NS is catalyzed by a 1,2-dioxygenase which weakens the carbon-sulfur bond.

Pusillimonas noertemannii gen. nov., sp. nov., a new member of the family Alcaligenaceae that degrades substituted salicylates The taxonomic position of a Pseudomonas-like strain, designated BN9 T , was investigated. This strain had previously been isolated as a 5-aminosalicylate-degrading organism from a 6-aminonaphthalene-2-sulphonate-degrading mixed bacterial culture. Previously, detection of ubiquinone Q-8, a polyamine pattern with putrescine, spermidine and 2-hydroxyputrescine as the major polyamines, and partial 16S rRNA gene sequencing had suggested that strain BN9 T belongs to the 'Betaproteobacteria'. This was supported by sequencing the 16S rRNA gene, which demonstrated 94-96 % sequence similarity to different species of the genera Achromobacter, Alcaligenes and Bordetella, and suggested that strain BN9 T represents a member of the family Alcaligenaceae. Different methods for the construction of phylogenetic dendrograms placed the strain separately from all other genera within the Alcaligenaceae. Fatty acid analysis demonstrated the presence of high concentrations of C 19 : 0 cyclo v8c. On the basis of low 16S rRNA gene sequence similarity to other members of the Alcaligenaceae, fatty acid and polar lipid profiles, and other unique phenotypic properties of strain BN9 T , the creation of a new genus and species with the name Pusillimonas noertemannii gen. nov., sp. nov. is proposed. The type strain is BN9 T

An extradiol dioxygenase was cloned from the naphthalenesulfonate-degrading bacterial strain BN6 by screening a gene bank for colonies with 2,3-dihydroxybiphenyl dioxygenase activity. DNA sequence analysis of a 1,358-bp fragment revealed an open reading frame of only 486 bp. This is the smallest gene encoding an extradiol dioxygenase found until now. Expression of the gene in a T7 expression vector enabled purification of the enzyme. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the protein was a dimer with a subunit size of 21.7 kDa. The enzyme oxidized 2,3-dihydroxybiphenyl, 3-isopropylcatechol, 3-and 4-chlorocatechol, and 3-and 4-methylcatechol. Since the ability to convert 3-chlorocatechol is an unusual characteristic for an extradiol-cleaving dioxygenase, this reaction was analyzed in more detail. The deduced amino-terminal amino acid sequence differed from the corresponding sequence of the 1,2-dihydroxynaphthalene dioxygenase, which had been determined earlier from the enzyme purified from this strain. This indicates that strain BN6 carries at least two different extradiol dioxygenases.A bacterial strain (strain BN6) which is able to degrade amino-and hydroxynaphthalenesulfonates is currently being studied in this laboratory. This strain oxidizes substituted naphthalenesulfonates to the corresponding substituted 1,2-dihydroxynaphthalenes by a reaction catalyzed by a desulfonating dioxygenase. Subsequent metabolism of the substituted 1,2-dihydroxynaphthalenes (1,2-DHNs) to substituted salicylates follows the common naphthalene degradative pathway. Strain BN6 does not mineralize naphthalenesulfonates, since the salicylates are not further oxidized (24,25,33,34). Cloning the genes for the metabolism of these naphthalenesulfonates would facilitate mobilization into salicylate-degrading bacterial strains, thereby accomplishing the complete degradation of naphthalenesulfonates in a single strain. Previously, the 1,2-DHN dioxygenase (DHNDO), which is part of this metabolic pathway, was purified to homogeneity and biochemically characterized. The enzyme oxidized 1,2-DHN, 2,3-dihydroxybiphenyl (2,3-DHBP), and some other aromatic diols (25). In the present study, we attempted to clone the corresponding gene from strain BN6. Although we obtained clones derived from strain BN6 which were able to oxidize 2,3-DHBP, the encoded gene was unexpectedly not involved in the degradation of naphthalenesulfonates. The extradiol dioxygenase (DO) encoded by this clone was examined in the present study. MATERIALS AND METHODSBacterial strains and culture conditions. The isolation and characterization of strain BN6 have been described previously (33). The strain has been deposited at the Deutsche Sammlung von Mikroorganismen, Brunswick, Germany, as DSM 6383. For the isolation of genomic DNA, the strain was grown in nutrient broth. Escherichia coli DH5␣ was the host for construction of the genomic library. E. coli JM 109 was used for subcloning and isolation of DNA for sequencing. Fo...

In cell extracts of Pseudaminobacter salicylatoxidans strain BN12, an enzymatic activity was detected which converted salicylate in an oxygen-dependent but NAD(P)H-independent reaction to a product with an absorbance maximum at 283 nm. This metabolite was isolated, purified, and identified by mass spectrometry and 1 H and 13 C nuclear magnetic resonance spectroscopy as 2-oxohepta-3,5-dienedioic acid. This metabolite could be formed only by direct ring fission of salicylate by a 1,2-dioxygenase reaction. Cell extracts from P. salicylatoxidans also oxidized 5-aminosalicylate, 3-, 4-, and 5-chlorosalicylate, 3-, 4-, and 5-methylsalicylate, 3-and 5-hydroxysalicylate (gentisate), and 1-hydroxy-2-naphthoate. The dioxygenase was purified and shown to consist of four identical subunits with a molecular weight of about 45,000. The purified enzyme showed higher catalytic constants with gentisate or 1-hydroxy-2-naphthoate than with salicylate. It was therefore concluded that P. salicylatoxidans synthesized a gentisate 1,2-dioxygenase with an extraordinary substrate range, which also allowed the oxidation of salicylate.Naphthalenesulfonic acids are produced on a large scale as industrial detergents, dispersive materials, and intermediates for the production of azo dyes (34). Some pure and mixed bacterial cultures which grow with naphthalene-1-sulfonic acid, naphthalene-2-sulfonic acid (2NS), 2-aminonaphthalene-1-sulfonic acid, 6-aminonaphthalene-2-sulfonic acid (6A2NS), or naphthalenedisulfonic acids as the sole source of carbon and energy have been isolated. The initial reaction in the microbial degradation of (substituted) naphthalenesulfonic acids is catalyzed by desulfonating dioxygenases, which catalyze the formation of (substituted) 1,2-dihydroxynaphthalene(s) and the release of sulfite (4, 5, 26-30, 32, 44). Thus, the degradative pathway for naphthalenesulfonic acids converges at the level of 1,2-dihydroxynaphthalene (1,2-DHN) with the well-known pathway for the degradation of naphthalene (Fig. 1). The reactions which are catalyzed by the "upper" naphthalenesulfonic acid pathway result in the formation of (substituted) salicylate(s). For Sphingomonas xenophaga BN6, it was found that the enzymes participating in the upper naphthalenesulfonic acid pathway convert a wide range of substituted substrates and that therefore a single set of enzymes is sufficient for the conversion of various substituted naphthalenesulfonic acids (19-21, 26, 27, 35). In contrast to the degradation of (substituted) naphthalenesulfonic acids to the corresponding salicylates, the productive metabolism of (substituted) salicylates requires rather different metabolic pathways. Thus, salicylate is usually converted by a salicylate 1-monooxygenase to catechol, 3-hydroxysalicylate (2,3-dihyroxybenzoate) is directly oxidized by an extradiol ring fission reaction to a nonaromatic product, 4-hydroxysalicylate (2,4-dihydroxybenzoate) is converted by a monooxygenase to the ring fission substrate 1,2,4-trihydroxybenzene, and gentisate (5-hydroxysalicylate...

intermedia, and Y. rohdei strains from Y. enterocolitica, which could not be differentiated by the API 20E test system. The probability for correct biotype identification of Y. enterocolitica isolates was 98.3% (41 externally validated strains). For correct serotype identification, the probability was 92.5% (42 externally validated strains). In addition, the presence or absence of the ail gene, one of the main pathogenicity markers, was demonstrated using FT-IR. The probability for correct identification of isolates concerning the ail gene was 98.5% (51 externally validated strains). This indicates that it is possible to obtain information about genus, species, and in the case of Y. enterocolitica also subspecies type with a single measurement. Furthermore, this is the first example of the identification of specific pathogenicity using FT-IR.