A second [2Fe-2S] ferredoxin from Sphingomonas sp. Strain RW1 can function as an electron donor for the dioxin dioxygenase

The first step in the degradation of dibenzofuran and dibenzo-p-dioxin by Sphingomonas sp. strain RW1 is carried out by dioxin dioxygenase (DxnA1A2), a ring-dihydroxylating enzyme. An open reading frame (fdx3) that could potentially specify a new ferredoxin has been identified downstream of dxnA1A2, a two-cistron gene (J. Armengaud, B. Happe, and K. N. Timmis, J. Bacteriol. 180:3954-3966, 1998). In the present study, we report a biochemical analysis of Fdx3 produced in Escherichia coli. This third ferredoxin thus far identified in Sphingomonas sp. strain RW1 contained a putidaredoxin-type [2Fe-2S] cluster which was characterized by UV-visible absorption spectrophotometry and electron paramagnetic resonance spectroscopy. The midpoint redox potential of this ferredoxin (E'(0) = -247 +/- 10 mV versus normal hydrogen electrode at pH 8.0) is similar to that exhibited by Fdx1 (-245 mV), a homologous ferredoxin previously characterized in Sphingomonas sp. strain RW1. In in vitro assays, Fdx3 can be reduced by RedA2 (a reductase similar to class I cytochrome P-450 reductases), previously isolated from Sphingomonas sp. strain RW1. RedA2 exhibits a K(m) value of 3.2 +/- 0.3 microM for Fdx3. In vivo coexpression of fdx3 and redA2 with dxnA1A2 confirmed that Fdx3 can serve as an electron donor for the dioxin dioxygenase.     

Identification and phenotypic characterization of Sphingomonas wittichii strain RW1 by peptide mass fingerprinting using matrix-assisted laser desorption ionization-time of flight mass spectrometry

Mass spectrometry is a potentially attractive means of monitoring the survival and efficacy of bioaugmentation agents, such as the dioxin-mineralizing bacterium Sphingomonas wittichii strain RW1. The biotransformation activity of RW1 phenotypes is determined primarily by the presence and concentration of the dioxin dioxygenase, an enzyme initiating the degradation of both dibenzo-p-dioxin and dibenzofuran (DF). We explored the possibility of identifying and characterizing putative cultures of RW1 by peptide mass fingerprinting (PMF) targeting this characteristic phenotypic biomarker. The proteome from cells of RW1--grown on various media in the presence and absence of DF--was partially purified, tryptically digested, and analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Mascot online database queries allowed statistically significant identification of RW1 in disrupted, digested cells (P < 0.01 to 0.05) and in digested whole-cell extracts (P < 0.00001 to 0.05) containing hundreds of proteins, as determined by two-dimensional gel electrophoresis. Up to 14 peptide ions of the alpha subunit of the dioxin dioxygenase (43% protein coverage) were detected in individual samples. A minimum of 10(7) DF-grown cells was required to identify dioxin degradation-enabled phenotypes. The technique hinges on the detection of multiple characteristic peptides of a biomarker that can reveal at once the identity and phenotypic properties of the microbial host expressing the protein. The results demonstrate the power of PMF of minimally processed microbial cultures as a sensitive and specific technique for the positive identification and phenotypic characterization of certain microorganisms used in biotechnology and bioremediation.     

Identification and Phenotypic Characterization of Sphingomonas wittichii Strain RW1 by Peptide Mass Fingerprinting Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Mass spectrometry is a potentially attractive means of monitoring the survival and efficacy of bioaugmentation agents, such as the dioxin-mineralizing bacterium Sphingomonas wittichii strain RW1. The biotransformation activity of RW1 phenotypes is determined primarily by the presence and concentration of the dioxin dioxygenase, an enzyme initiating the degradation of both dibenzo-p-dioxin and dibenzofuran (DF). We explored the possibility of identifying and characterizing putative cultures of RW1 by peptide mass fingerprinting (PMF) targeting this characteristic phenotypic biomarker. The proteome from cells of RW1—grown on various media in the presence and absence of DF—was partially purified, tryptically digested, and analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Mascot online database queries allowed statistically significant identification of RW1 in disrupted, digested cells (P < 0.01 to 0.05) and in digested whole-cell extracts (P < 0.00001 to 0.05) containing hundreds of proteins, as determined by two-dimensional gel electrophoresis. Up to 14 peptide ions of the alpha subunit of the dioxin dioxygenase (43% protein coverage) were detected in individual samples. A minimum of 10⁷ DF-grown cells was required to identify dioxin degradation-enabled phenotypes. The technique hinges on the detection of multiple characteristic peptides of a biomarker that can reveal at once the identity and phenotypic properties of the microbial host expressing the protein. The results demonstrate the power of PMF of minimally processed microbial cultures as a sensitive and specific technique for the positive identification and phenotypic characterization of certain microorganisms used in biotechnology and bioremediation.     

Bacterial degradation of aromatic compounds via angular dioxygenation

Dioxygenation is one of the important initial reactions of the bacterial degradation of various aromatic compounds. Aromatic compounds, such as biphenyl, toluene, and naphthalene, are dioxygenated at lateral positions of the aromatic ring resulting in the formation of cis-dihydrodiol. This "normal" type of dioxygenation is termed lateral dioxygenation. On the other hand, the analysis of the bacterial degradation of fluorene (FN) analogues, such as 9-fluorenone, dibenzofuran (DF), carbazole (CAR), and dibenzothiophene (DBT)-sulfone, and DF-related diaryl ether compounds, dibenzo-p-dioxin (DD) and diphenyl ether (DE), revealed the presence of the novel mode of dioxygenation reaction for aromatic nucleus, generally termed angular dioxygenation. In this atypical dioxygenation, the carbon bonded to the carbonyl group in 9-fluorenone or to heteroatoms in the other compounds, and the adjacent carbon in the aromatic ring are both oxidized. Angular dioxygenation of DF, CAR, DBT-sulfone, DD, and DE produces the chemically unstable hemiacetal-like intermediates, which are spontaneously converted to 2,2',3-trihydroxybiphenyl, 2'-aminobiphenyl-2,3-diol, 2',3'-dihydroxybiphenyl-2-sulfinate, 2,2',3-trihydroxydiphenyl ether, and phenol and catechol, respectively. Thus, angular dioxygenation for these compounds results in the cleavage of the three-ring structure or DE structure. The angular dioxygenation product of 9-fluorenone, 1-hydro-1,1a-dihydroxy-9-fluorenone is a chemically stable cis-diol, and is enzymatically transformed to 2'-carboxy-2,3-dihydroxybiphenyl. 2'-Substituted 2,3-dihydroxybiphenyls formed by angular dioxygenation of FN analogues are degraded to monocyclic aromatic compounds by meta cleavage and hydrolysis. Thus, after the novel angular dioxygenation, subsequent degradation pathways are homologous to the corresponding part of that of biphenyl. Compared to the bacterial strains capable of catalyzing lateral dioxygenation, few bacteria having angular dioxygenase have been reported. Only a few degradation pathways, CAR-degradation pathway of Pseudomonas resinovorans strain CA10, DF/DD-degradation pathway of Sphingomonas wittichii strain RW1, DF/DD/FN-degradation pathway of Terrabacter sp. strain DBF63, and carboxylated DE-degradation pathway of P. pseudoalcaligenes strain POB310, have been investigated at the gene level. As a result of the phylogenetic analysis and the comparison of substrate specificity of angular dioxygenase, it is suggested that this atypical mode of dioxygenation is one of the oxygenation reactions originating from the relaxed substrate specificity of the Rieske nonheme iron oxygenase superfamily. Genetic characterization of the degradation pathways of these compounds suggests the possibility that the respective genetic elements constituting the entire catabolic pathway have been recruited from various other bacteria and/or other genetic loci, and that these pathways have not evolutionary matured.     

Degradation of Chlorinated Dibenzofurans and Dibenzo-p-Dioxins by Sphingomonas sp. Strain RW1

The ability of the dibenzofuran- and dibenzo-p-dioxin-mineralizing bacterium Sphingomonas sp. strain RW1 (R.-M. Wittich, H. Wilkes, V. Sinnwell, W. Francke, and P. Fortnagel, Appl. Environ. Microbiol. 58:1005-1010, 1992) to oxidize chlorinated derivatives of dibenzofuran and dibenzo-p-dioxin was analyzed. Strain RW1 degraded several mono- and dichlorinated dibenzofurans and dibenzo-p-dioxins, but it did not degrade more highly chlorinated congeners. Most mono- and dichlorinated dibenzofurans and dibenzo-p-dioxins investigated in this study were degraded to the corresponding mono- and dichlorinated salicylates and catechols, respectively, together with salicylate and catechol. This indicates an initial dioxygenolytic attack on the substituted as well as on the nonsubstituted aromatic nucleus of most of the target compounds. Strain RW1 could not grow at the expense of monochlorinated dibenzo-p-dioxins and dibenzofurans as carbon sources, with the exception of 4-chlorodibenzofuran, which was stoichiometrically converted to 3-chlorosalicylate.     

A Functional 4-Hydroxysalicylate/Hydroxyquinol Degradative Pathway Gene Cluster Is Linked to the Initial Dibenzo-p-Dioxin Pathway Genes in Sphingomonas sp. Strain RW1

The bacterium Sphingomonas sp. strain RW1 is able to use dibenzo-p-dioxin, dibenzofuran, and several hydroxylated derivatives as sole sources of carbon and energy. We have determined and analyzed the nucleic acid sequence of a 9,997-bp HindIII fragment downstream of cistrons dxnA1A2, which encode the dioxygenase component of the initial dioxygenase system of the corresponding catabolic pathways. This fragment contains 10 colinear open reading frames (ORFs), apparently organized in one compact operon. The enzymatic activities of some proteins encoded by these genes were analyzed in the strain RW1 and, after hyperexpression, in Escherichia coli. The first three ORFs of the locus, designated dxnC, ORF2, and fdx3, specify a protein with a low homology to bacterial siderophore receptors, a polypeptide representing no significant homology to known proteins, and a putative ferredoxin, respectively. dxnD encodes a 69-kDa phenol monooxygenase-like protein with activity for the turnover of 4-hydroxysalicylate, and dxnE codes for a 37-kDa protein whose sequence and activity are similar to those of known maleylacetate reductases. The following gene, dxnF, encodes a 33-kDa intradiol dioxygenase which efficiently cleaves hydroxyquinol, yielding maleylacetate, the ketoform of 3-hydroxy-cis,cis-muconate. The heteromeric protein encoded by dxnGH is a 3-oxoadipate succinyl coenzyme A (succinyl-CoA) transferase, whereas dxnI specifies a protein exhibiting marked homology to acetyl-CoA acetyltransferases (thiolases). The last ORF of the sequenced fragment codes for a putative transposase. DxnD, DxnF, DxnE, DxnGH, and DxnI (the activities of most of them have also been detected in strain RW1) thus form a complete 4-hydroxysalicylate/hydroxyquinol degradative pathway. A route for the mineralization of the growth substrates 3-hydroxydibenzofuran and 2-hydroxydibenzo-p-dioxin in Sphingomonas sp. strain RW1 thus suggests itself.     

Isolation and characterization of the genes encoding a novel oxygenase component of angular dioxygenase from the gram-positive dibenzofuran-degrader Terrabacter sp. strain DBF63

A gram-positive bacterium Terrabacter sp. strain DBF63 is able to degrade dibenzofuran (DF) via initial dioxygenation by a novel angular dioxygenase. The dbfA1 and dbfA2 genes, which encode the large and small subunits of the dibenzofuran 4,4a-dioxygenase (DFDO), respectively, were isolated by a polymerase chain reaction-based method. DbfA1 and DbfA2 showed moderate homology to the large and small subunits of other ring-hydroxylating dioxygenases (less than 40%), respectively, and some motifs such as the Fe(II) binding site and the [2Fe-2S] cluster ligands were conserved in DbfA1. DFDO activity was confirmed in Escherichia coli cells containing the cloned dbfA1 and dbfA2 genes with the complementation of nonspecific ferredoxin and ferredoxin reductase component of E. coli. Under this condition, these cells exhibited angular dioxygenation of DF and dibenzo-p-dioxin, and monooxygenation of fluorene, but not angular dioxygenation of carbazole, xanthene, and phenoxathiin. Phylogenetic analysis revealed that DbfA1 formed a branch with recently reported large subunits of polycyclic aromatic hydrocarbon (PAH) dioxygenase from gram-positive bacteria but did not cluster with that of other angular dioxygenases, i.e., DxnA1 from Sphingomonas sp. strain RW1 [Armengaud, J., Happe, B., and Timmis, K. N. J. Bacteriol. 180, 3954-3966, 1998] and CarAa from Pseudomonas sp. strain CA10 [Sato, S., Nam, J.-W., Kasuga, K., Nojiri, H., Yamane, H., and Omori, T. J. Bacteriol. 179, 4850-4858, 1997].     

Superior survival and degradation of dibenzo-p-dioxin and dibenzofuran in soil by soil-adapted Sphingomonas sp. strain RW1

The dibenzo-p-dioxin(DD)- and dibenzofuran(DF)-degrading bacterium, Sphingomonas sp. strain RW1, was tagged by insertion of a mini-Tn5 lacZ transposon in order to follow its fate in complex laboratory soil systems. The tagged strain was tested for its ability to survive in soil and degrade DF and DD applied at a concentration of 1 mg/g. Bacteria pre-adapted to soil conditions were found to survive better in DF- and DD-amended soil and degrade the substrate more efficiently than bacteria that had not been subjected to pre-adaptation. The concentration of soil-applied DF and DD, individually and in combination, decreased to less than 2% of the original concentrations within 3 weeks of addition of the RW1 derivative, accompanied by a short, but significant exponential increase in RW1 viable cells. During the same period the native bacterial population in soil was stable while viable fungi declined. Received: 12 November 1996 / Received revision: 21 February 1997 / Accepted: 22 February 1997     

Conversion of chlorobiphenyls into phenylhexadienoates and benzoates by the enzymes of the upper pathway for polychlorobiphenyl degradation encoded by the bph locus of Pseudomonas sp. strain LB400.

Metabolism of 21 chlorobiphenyls by the enzymes of the upper biphenyl catabolic pathway encoded by the bph locus of Pseudomonas sp. strain LB400 was investigated by using recombinant strains harboring gene cassettes containing bphABC or bphABCD. The enzymes of the upper pathway were generally able to metabolize mono- and dichlorinated biphenyls but only partially transform most trichlorinated congeners investigated: 14 of 15 mono- and dichlorinated and 2 of 6 trichlorinated congeners were converted into benzoates. All mono- and at least 8 of 12 dichlorinated congeners were attacked by the bphA-encoded biphenyl dioxygenase virtually exclusively at ortho and meta carbons. This enzyme exhibited a high degree of selectivity for the aromatic ring to be attacked, with the order of ring preference being non- > ortho- > meta- > para-substituted for mono- and dichlorinated congeners. The influence of the chlorine substitution pattern of the metabolized ring on benzoate formation resembled its influence on the reactivity of initial dioxygenation, suggesting that the rate of benzoate formation may frequently be determined by the rate of initial attack. The absorption spectra of phenylhexadienoates formed correlated with the presence or absence of a chlorine substituent at an ortho position.     

Detection and characterization of conjugative degradative plasmids in xenobiotic-degrading Sphingomonas strains

A systematic survey for the presence of plasmids in 17 different xenobiotic-degrading Sphingomonas strains was performed. In almost all analyzed strains, two to five plasmids with sizes of about 50 to 500 kb were detected by using pulsed-field gel electrophoresis. A comparison of plasmid preparations untreated or treated with S1 nuclease suggested that, in general, Sphingomonas plasmids are circular. Hybridization experiments with labeled gene probes suggested that large plasmids are involved in the degradation of dibenzo-p-dioxin, dibenzofuran, and naphthalenesulfonates in S. wittichii RW1, Sphingomonas sp. HH69, and S. xenophaga BN6, respectively. The plasmids which are responsible for the degradation of naphthalene, biphenyl, and toluene by S. aromaticivorans F199 (pNL1) and of naphthalenesulfonates by S. xenophaga BN6 (pBN6) were site-specifically labeled with a kanamycin resistance cassette. The conjugative transfer of these labeled plasmids was attempted with various bacterial strains as putative recipient strains. Thus, a conjugative transfer of plasmid pBN6 from S. xenophaga BN6 to a cured mutant of strain BN6 and to Sphingomonas sp. SS3 was observed. The conjugation experiments with plasmid pNL1 suggested a broader host range of this plasmid, because it was transferred without any obvious structural changes to S. yanoikuyae B1, Sphingomonas sp. SS3, and S. herbicidovorans. In contrast, major plasmid rearrangements were observed in the transconjugants after the transfer of plasmid pNL1 to Sphingomonas sp. HH69 and of pBN6 to Sphingomonas sp. SS3. No indications for the transfer of a Sphingomonas plasmid to bacteria outside of the Sphingomonadaceae were obtained.     

Genetic characterization of the dibenzofuran-degrading Actinobacteria carrying the dbfA1A2 gene homologues isolated from activated sludge

Thirteen dibenzofuran (DF)-utilizing bacteria carrying the DF terminal dioxygenase genes homologous to those of Terrabacter sp. strain DBF63 (dbfA1A2) were newly isolated from activated sludge samples. The amplified ribosomal DNA restriction analysis and the hybridization analyses showed that these strains were grouped into five genetically different types of bacteria. The sequence analyses of the 16S rRNA genes and the dbfA1A2 homologues from these five selected isolates revealed that the isolates belonged to the genus Rhodococcus, Terrabacter or Janibacter and that they shared 99-100% conserved dbfA1A2 homologues. We investigated the genetic organizations flanking the dbfA1A2 homologues and showed that the minimal conserved DNA region present in all five selected isolates consisted of an approximately 9.0-kb region and that their outer regions became abruptly non-homologous. Among them, Rhodococcus sp. strain DFA3 possessed not only the 9.0-kb region but also the 6.2-kb region containing dbfA1A2 homologues. Sequencing of their border regions suggested that some genetic rearrangement might have occurred with insertion sequence-like elements. Also, within their conserved regions, some insertions or deletions were observed.     

Isolation and characterization of dibenzofuran-degrading actinomycetes: analysis of multiple extradiol dioxygenase genes in dibenzofuran-degrading Rhodococcus species

Sixteen actinomycetes capable of utilizing dibenzofuran as a sole source of carbon and energy were isolated, including Rhodococcus, Microbacterium, and Terrabacter genera. Heretofore, no dibenzofuran-utilizing strain belonging to the genus Microbacterium has been reported. Five extradiol dioxygenase genes (dfdB, and edil to 4) of the strain Rhodococcus sp. YK2 were cloned and analyzed. The nucleotide sequence of dfdB gene was almost identical to the bphC1 gene of Terrabacter sp. DPO360, which was involved in dibenzofuran metabolism in this strain. Southern and Northern hybridization analyses using these extradiol dioxygenase genes as probes suggest that the dfdB gene in YK2 was conserved in diverse dibenzofuran-utilizing actinomycetes; also, the dfdB gene was the only expressed gene among five extradiol dioxygenase genes in the medium containing DF as a sole carbon source. These results suggest that the dfdB gene is important for dibenzofuran metabolism not only in the strain YK2, but also in diverse dibenzofuran-degrading actinomycetes.     

Isolation and characterization of a gene cluster for dibenzofuran degradation in a new dibenzofuran-utilizing bacterium, Paenibacillus sp. strain YK5

Spore-forming bacterial strains capable of utilizing dibenzofuran (DF) as a sole source of carbon and energy were isolated. Characteristics of the isolates justified their classification into the genus Paenibacillus, and their closest relative was P. naphthalenovorans. Degenerate primers for aromatic hydrocarbon dioxygenase alpha subunit (AhDOa) genes and genomic DNA of the strain YK5 were used for gene isolation. The nucleotide sequences of clones of the PCR products revealed that the strain YK5 carries at least five different AhDOa genes. Northern hybridization analysis showed that one of the AhDOa genes was transcribed under DF-containing culture conditions. A gene cluster encoding the AhDOa was isolated. The genes predicted to encode extradiol dioxygenase (dbfB) and hydrolase (dbfC) were found to be an upstream of genes encoding the alpha and beta subunit of the AhDO (dbfA1 and dbfA2, respectively); the latter two gene products showed 60 and 53% identity to the amino acid sequences of DbfA1 and DbfA2 of Terrabacter sp. DBF63, respectively. Two Paenibacillus validus JCM 9077 strains transformed with the dbf gene clusters acquired the ability to convert DF to 2,2′,3-trihydroxybiphenyl (THBP) and salicylic acid (SAL). These results suggest that the enzymes encoded by the gene cluster isolated in this study are involved in DF metabolism in YK5.     

Molecular detection and diversity of polycyclic aromatic hydrocarbon-degrading bacteria isolated from geographically diverse sites

Nineteen polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were isolated from environmental samples in Kuwait, Indonesia, Thailand, and Japan by enrichment with either naphthalene or phenanthrene as a sole carbon source. Sequence analyses of the 16-S rRNA gene indicated that at least seven genera (Ralstonia, Sphingomonas, Burkholderia, Pseudomonas, Comamonas, Flavobacterium, and Bacillus) were present in this collection. Determination of the ability of the isolates to use PAH and its presumed catabolic intermediates suggests that the isolates showed multiple phenotypes in terms of utilization and degradation pathways. The large subunit of the terminal oxygenase gene (phnAc) from Burkholderia sp. strain RP007 hybridized to 32% (6/19) of the isolates, whilst gene probing using the large subunit of terminal oxygenase gene (pahAc) from Pseudomonas putida strain OUS82 revealed no pahAc-like genes amongst the isolates. Using three degenerated primer sets (pPAH-F/NR700, AJ025/26, and RieskeF/R), targeting a conserved region with the genes encoding the large subunit of terminal oxygenase successfully amplified material from 6 additional PAH-degrading isolates. Sequence analyses showed that the large subunit of terminal oxygenase in 4 isolates was highly homologous to the large subunit of naphthalene dioxygenase gene from Ralstonia sp. strain U2. However, we could not obtain any information on the oxygenase system involved in the naphthalene and/or phenathrene degradation by 7 other strains. These results suggest that PAH-degrading bacteria are diverse, and that there are still many unidentified PAH-degrading bacteria.