Toshio Omori

Plasmid pCAR3 Contains Multiple Gene Sets Involved in the Conversion of Carbazole to Anthranilate

ABSTRACT The carbazole degradative car-I gene cluster (carAaIBaIBbICIAcI) of Sphingomonas sp. strain KA1 is located on the 254-kb circular plasmid pCAR3. Carbazole conversion to anthranilate is catalyzed by carbazole 1,9a-dioxygenase (CARDO; CarAaIAcI), meta-cleavage enzyme (CarBaIBbI), and hydrolase (CarCI). CARDO is a three-component dioxygenase, and CarAaI and CarAcI are its terminal oxygenase and ferredoxin components. The car-I gene cluster lacks the gene encoding the ferredoxin reductase component of CARDO. In the present study, based on the draft sequence of pCAR3, we found multiple carbazole degradation genes dispersed in four loci on pCAR3, including a second copy of the car gene cluster (carAaIIBaIIBbIICIIAcII) and the ferredoxin/reductase genes fdxI-fdrI and fdrII. Biotransformation experiments showed that FdrI (or FdrII) could drive the electron transfer chain from NAD(P)H to CarAaI (or CarAaII) with the aid of ferredoxin (CarAcI, CarAcII, or FdxI). Because this electron transfer chain showed phylogenetic relatedness to that consisting of putidaredoxin and putidaredoxin reductase of the P450cam monooxygenase system of Pseudomonas putida, CARDO systems of KA1 can be classified in the class IIA Rieske non-heme iron oxygenase system. Reverse transcription-PCR (RT-PCR) and quantitative RT-PCR analyses revealed that two car gene clusters constituted operons, and their expression was induced when KA1 was exposed to carbazole, although the fdxI-fdrI and fdrII genes were expressed constitutively. Both terminal oxygenases of KA1 showed roughly the same substrate specificity as that from the well-characterized carbazole degrader Pseudomonas resinovorans CA10, although slight differences were observed.

Novel marine carbazole-degrading bacteria

Eleven carbazole (CAR)-degrading bacterial strains were isolated from seawater collected off the coast of Japan using two different media. Seven isolates were shown to be most closely related to the genera Erythrobacter, Hyphomonas, Sphingosinicella, Caulobacter, and Lysobacter. Meanwhile, strains OC3, OC6S, OC9, and OC11S showed low similarity to known bacteria, the closest relative being Kordiimonas gwangyangensis GW14-5 (90% similarity). Southern hybridization analysis revealed that only five isolates carried car genes similar to those reported in Pseudomonas resinovorans CA10 (car(CA10)) or Sphingomonas sp. strain KA1 (car(KA1)). The isolates were subjected to GC-MS and the results indicated that these strains degrade CAR to anthranilic acid.

Differentiation of Carbazole Catabolic Operons by Replacement of the Regulated Promoter via Transposition of an Insertion Sequence

The carbazole catabolic car operons from Pseudomonas resinovorans CA10 and Janthinobacterium sp. J3 have nearly identical nucleotide sequences in their structural and intergenic regions but not in their flanking regions. Transposition of ISPre1 from the anthranilate catabolic ant operon located an inducible promoter Pant upstream of the carCA10 operon, which is regulated by the AraC/XylS family activator AntR in response to anthranilate. The transposed Pant drives transcription of the carCA10 operon, which is composed of the car-AaAaBaBbCAcAdDFECA10 structural genes. Transcriptional fusion truncating Pant upstream of carAaCA10 resulted in constitutive luciferase expression. Primer extension analysis identified a transcription start point of the constitutive mRNA of the carCA10 operon at 385 nucleotides upstream of the carAaCA10 translation start point, and the PcarAa promoter was found. On the other hand, a GntR family regulatory gene carRJ3 is divergently located upstream of the carJ3 operon. The Pu13 promoter, required for inducible transcription of the carJ3 operon in the presence of carbazole, was identified in the region upstream of carAaJ3, which had been replaced with the Pant promoter in the carCA10 operon. Deletion of carRJ3 from a transcriptional fusion resulted in high level constitutive expression from Pu13. Purified CarRJ3 protein bound at two operator sequences OI and OII, showing that CarRJ3 directly represses Pu13 in the absence of its inducer, which was identified as 2-hydroxy-6-oxo-6-(2′-aminophenyl)hexa-2,4-dienoate, an intermediate of the carbazole degradation pathway.

Isolation and characterization of a car gene cluster from the naphthalene, phenanthrene, and carbazole-degrading marine isolate Lysobacter sp. strain OC7.

The novel carbazole (CAR)-degrading bacterium Lysobacter sp. strain OC7 has been isolated from seawater and can also utilize naphthalene and phenanthrene as its sole carbon and energy source. The CAR-degradative gene cluster was isolated and encoded five complete open reading frames (ORFs) and two truncated ORFs. Among them, four ORFs showed 40–50% similarity with previously reported CAR-degradative genes. Ferredoxin (carAc) and ferredoxin reductase (carAd) genes, which are necessary for the CAR 1,9a-dioxygenase system, were not found in this car gene cluster. The carOC7 gene transcripts were strongly detected when CAR was provided. However, these transcripts were also detected when naphthalene was provided. The resting cell reaction with Escherichia coli revealed that CarAaOC7 can use CarAc and CarAd of Pseudomonas resinovorans CA10 as ferredoxin and ferredoxin reductase, respectively, and converted CAR to 2′-aminobiphenyl-2,3-diol. In 13 marine CAR-degrading isolates, only Caulobacter sp. strain OC6 hybridized with the carOC7 gene cluster probe. This is the first report showing CAR-degradative genes from the genus Lysobacter.

Carbazole Metabolism by Pseudomonads

Abbreviations: BDO, biphenyl 2,3-dioxygenase; BDO-F, ferredoxin component of BDO; bp, base pairs; CARDO, carbazole 1,9a-dioxygenase; CARDO-F, ferredoxin component of CARDO; CARDO-O, terminal oxygenase component of CARDO; CARDO-R, reductase component of CARDO; [2Fe–2S]P, plant-type [2Fe–2S] cluster; [2Fe–2S]Pu, putidaredoxin-type [2Fe–2S] cluster; [2Fe–2S]R, Rieske [2Fe–2S] cluster; HOADA, 2-hydroxy-6-oxo-6-(2′-aminophenyl)-hexa-2,4-dienoic acid; HPD, 2-hydroxypenta-2,4-dienoate; Inc, incompatibility; IR, inverted repeat; IS, insertion sequence; kb, kilobase; NDO, naphthalene 1,2-dioxygenase; NDO-O, terminal oxygenase component of NDO; OMO, 2-oxoquinoline 8-monooxygenase; OMO-O, terminal oxygenase component of OMO; ORF, open reading frame; rmsd, root mean square deviation; ROS, Rieske nonheme iron oxygenase system; TCA, tricarboxylic acid.

Cloning and nucleotide sequences of carbazole degradation genes from marine bacterium Neptuniibacter sp. strain CAR-SF

The marine bacterium Neptuniibacter sp. strain CAR-SF utilizes carbazole as its sole carbon and nitrogen sources. Two sets of clustered genes related to carbazole degradation, the upper and lower pathways, were obtained. The marine bacterium genes responsible for the upper carbazole degradation pathway, carAa, carBa, carBb, and carC, encode the terminal oxygenase component of carbazole 1,9a-dioxygenase, the small and large subunits of the meta-cleavage enzyme, and the meta-cleavage compound hydrolase, respectively. The genes involved in the lower degradation pathway encode the anthranilate dioxygenase large and small subunit AntA and AntB, anthranilate dioxygenase reductase AntC, 4-oxalocrotonate tautomerase, and catechol 2,3-dioxygenase. Reverse transcription-polymerase chain reaction confirmed the involvement of the isolated genes in carbazole degradation. Escherichia coli cells transformed with the CarAa of strain CAR-SF required ferredoxin and ferredoxin reductase for biotransformation of carbazole. Although carAc, which encodes the ferredoxin component of carbazole 1,9a-dioxygenase, was not found immediately downstream of carAaBaBbC, the carAc-like gene may be located elsewhere based on Southern hybridization. This is the first report of genes involved in carbazole degradation isolated from a marine bacterium.

Isolation and characterization of the gene encoding the chloroplast-type ferredoxin component of carbazole 1,9a-dioxygenase from a putative Kordiimonas sp.

Carbazole (CAR)-degrading genes (carRAaCBaBb) were isolated from marine CAR-degrading isolate strain OC9 (probably Kordiimonas gwangyangensis) using shotgun cloning experiments and showed 35–65% similarity with previously reported CAR-degrading genes. In addition, a ferredoxin-like gene (carAc) was found downstream of carR, although it was not homologous with any reported ferredoxin components of the CAR 1,9a-dioxygenase (CARDO) system. The carAc-deduced amino acid sequence possessed consensus sequences for chloroplast-type iron-sulfur proteins for binding the [2Fe-2S] cluster. These car genes were arranged in the order of carAcRAaCBaBb, but carRAc and carAaCBaBb genes were the opposite orientation. Escherichia coli JM109 cells harboring pBOC91 (carAa) converted CAR to 2′-aminobiphenyl-2,3-diol at a ratio of 12%, and the transformation ratio of CAR increased from 12 to 100% when carAc was added, indicating that CarAc is the ferredoxin component of the CARDO system in strain OC9. This is the first finding of a chloroplast-type ferredoxin component in a CARDO system. Biotransformation tests with aromatic compounds revealed that the strain OC9 CarAaAc showed activity with polycyclic aromatic hydrocarbons and dioxin compounds and exhibited significant activity for fluorene, unlike previously reported CARDOs.

A Series of Crystal Structures of a meta-Cleavage Product Hydrolase from Pseudomonas fluorescens IP01 (CumD) Complexed with Various Cleavage Products

Meta-cleavage product hydrolase (MCP-hydrolase) is one of the key enzymes in the microbial degradation of aromatic compounds. MCP-hydrolase produces 2-hydroxypenta-2,4-dienoate and various organic acids, according to the C6 substituent of the substrate. Comprehensive analysis of the substrate specificity of the MCP-hydrolase from Pseudomonas fluorescens IP01 (CumD) was carried out by determining the kinetic parameters for nine substrates and crystal structures complexed with eight cleavage products. CumD preferred substrates with long non-branched C6 substituents, but did not effectively hydrolyze a substrate with a phenyl group. Superimposition of the complex structures indicated that benzoate was bound in a significantly different direction than other aliphatic cleavage products. The directions of the bound organic acids appeared to be related with the k cat values of the corresponding substrates. The Ile139 and Trp143 residues on helix α4 appeared to cause steric hindrance with the aromatic ring of the substrate, which hampers base-catalyzed attack by water.

Ammonia accumulation in culture broth by the novel nitrogen-fixing bacterium, Lysobacter sp. E4

This is the first report that Lysobacter fixes nitrogen under free-living conditions, as shown by its ability to grow on nitrogen-free medium and accumulate relatively high amounts of ammonia in the culture broth. Growth of the E4 Lysobacter strain, isolated in a screen for nitrogen-fixing and ammonia-producing bacteria, resulted in higher ammonia accumulation (0.53 mM ammonium ion concentration) in media containing glucose rather than other tested carbon sources. The optimum glucose concentration was 0.30% at an initial medium pH of 7.0 and incubation temperature of 30°C. From time-course experiments, when the glucose in the culture was exhausted, ammonia began to be accumulated, and maximum ammonia accumulation (∼1.60 mM) was reached after 8 days of incubation. Ammonia accumulation by this strain required molybdenum, manganese, and iron.

Cloning of dfdA genes from Terrabacter sp. strain DBF63 encoding dibenzofuran 4,4a-dioxygenase and heterologous expression in Streptomyces lividans

A dibenzofuran (DF)-degrader Terrabacter sp. strain DBF63 harbors the dbfA and dbfBC genes for DF degradation and the fln-dbfA, pht, and pca gene clusters for the utilization of fluorene (FN) as a sole carbon source. From this strain, dfdA1, the gene encoding the second DF dioxygenase was detected using degenerate polymerase chain reaction (PCR) and the dfdA1A2A3A4 genes were cloned from a cosmid library of the DBF63 genome. Nucleotide sequencing revealed that the dfdA genes showed considerably high identities with those of other actinobacteria, such as Terrabacter sp. strain YK3 and Rhodococcus sp. strain HA01. In the neighboring region of the dfdA genes, as many as 11 homologs for transposase and integrase genes and the putative extradiol dioxygenase gene disrupted by an insertion sequence (dfdB::ISTesp2) were found, suggesting that repeated gene rearrangement had occurred. Quantitative reverse transcription-PCR analysis revealed that dfdA1 was expressed primarily in the DF-fed strain, whereas dbfA1 was expressed in the FN-cultured strain, apparently indicating that the dfdA genes are induced by DF for the initial hydroxylation of DF in strain DBF63. Furthermore, two polycistronic gene cassettes consisting of either dfdA or dbfA together with the dbfBC gene were constructed and expressed heterologously in Streptomyces lividans, degrading DF to salicylate. Furthermore, the expressed DfdA dioxygenase degraded dibenzo-p-dioxin, carbazole, dibenzothiophene, anthracene, phenanthrene, and biphenyl, thereby exhibiting a broader substrate range than that of the DbfA dioxygenase.