Genetic characterization and evolutionary implications of a car gene cluster in the carbazole degrader Pseudomonas sp. strain CA10

Abstract

The nucleotide sequences of the 27,939-bp-long upstream and 9,448-bp-long downstream regions of the carAaAaBaBbCAc(ORF7)Ad genes of carbazole-degrading Pseudomonas sp. strain CA10 were determined. Thirty-two open reading frames (ORFs) were identified, and the car gene cluster was consequently revealed to consist of 10 genes (carAaAaBaBbCAcAdDFE) encoding the enzymes for the three-step conversion of carbazole to anthranilate and the degradation of 2-hydroxypenta-2,4-dienoate. The high identities (68 to 83%) with the enzymes involved in 3-(3-hydroxyphenyl)propionic acid degradation were observed only for CarFE. This observation, together with the fact that two ORFs are inserted between carD and carFE, makes it quite likely that the carFE genes were recruited from another locus. In the 21-kb region upstream from carAa, aromatic-ring-hydroxylating dioxygenase genes (ORF26, ORF27, and ORF28) were found. Inductive expression in carbazole-grown cells and the results of homology searching indicate that these genes encode the anthranilate 1,2-dioxygenase involved in carbazole degradation. Therefore, these ORFs were designated antABC. Four homologous insertion sequences, IS5car1 to IS5car4, were identified in the neighboring regions of car and ant genes. IS5car2 and IS5car3 constituted the putative composite transposon containing antABC. One-ended transposition of IS5car2 together with the 5' portion of antA into the region immediately upstream of carAa had resulted in the formation of IS5car1 and ORF9. In addition to the insertion sequence-dependent recombination, gene duplications and presumed gene fusion were observed. In conclusion, through the above gene rearrangement, the novel genetic structure of the car gene cluster has been constructed. In addition, it was also revealed that the car and ant gene clusters are located on the megaplasmid pCAR1.

FIG. 1

Degradation pathway of CAR in Pseudomonas sp. strain CA10. Enzyme designations: CarAaAcAd, carbazole 1,9a-dioxygenase; CarBaBb, 2′-aminobiphenyl-2,3-diol 1,2-dioxygenase; CarC, 2-hydroxy-6-oxo-6-(2′-aminophenyl)-hexa-2,4-dienoic acid (meta-cleavage compound) hydrolase; CarD, 2-hydroxypenta-2,4-dienoate hydratase; CarE, 4-hydroxy-2-oxovalerate aldolase; CarF, acetaldehyde dehydrogenase (acylating); AntABC, anthranilate 1,2-dioxygenase; CatA, catechol 1,2-dioxygenase; CatB, cis,cis-muconate lactonizing enzyme; CatC, muconolactone δ-isomerase. Compounds: I, CAR; II, 2′-aminobiphenyl-2,3-diol; III, 2-hydroxy-6-oxo-6-(2′-aminophenyl)-hexa-2,4-dienoic acid (meta-cleavage compound); IV, anthranilic acid; V, catechol; VI, cis,cis-muconic acid; VII, muconolactone; VIII, β-ketoadipic acid enol-lactone; IX, 2-hydroxypenta-2,4-dienoic acid; X, 4-hydroxy-2-oxovaleric acid; XI, pyruvic acid; XII, acetaldehyde; XIII, acetyl coenzyme A.

FIG. 2

Physical map of the 44.3-kb DNA region containing the CAR-degrading car gene cluster. The pentagons and triangles in the physical map indicate the sizes, locations, and directions of transcription of the ORFs derived from the DNA sequence analysis. The shaded pentagons and triangles represent the car and ant gene clusters. The shaded boxes containing hatched pentagons represent the ISs, and the hatched pentagons indicate the sizes, locations, and directions of transcription of the transposase genes. The location of the subcloned DNA fragments and orientation of the lac promoter of the cloning vector are indicated by the lengths and the directions of the arrows, respectively. As for the cosmid clones, the DNA region which was revealed to be contained in the respective cosmids by Southern hybridization analysis is shown by the solid line. The double slash attached to the end of the solid line indicates that the DNA region outside the EcoRI site was contained in the respective cosmid insert. Only the restriction enzyme sites used for the construction of the plasmids are shown. Restriction enzyme site designations: C, ClaI; EI, EcoRI; EV, EcoRV; Hc, HincII; Hd, HindIII; P, PstI; Sl, SalI; Sp, SphI; Xb, XbaI; and Xh, XhoI.

FIG. 3

Alignments of TnpA1 and TnpA3 identified from strain CA10 with their homologous transposases. The aligned transposases are contained in ISs classified in the IS5 family (32). The identical and similar amino acid residues in all proteins are indicated by asterisks and dots, respectively. The gaps introduced into the alignment are indicated by hyphens. The critical DDE consensus motif and the well-conserved R (or K) residue 7 amino acids downstream from the E residue in N2, N3, and C1 domains (32) are indicated by the white type. The DDBJ, EMBL, and GenBank nucleotide sequence accession numbers are indicated after the names of transposases and the names of ISs or bacterial strains (in parentheses).

FIG. 4

Alignments of the large (α) subunits of class I terminal oxygenase components with the postulated ORF18 and antA gene products. The identical and similar amino acid residues in all proteins are indicated by asterisks and dots, respectively. The gaps introduced into the alignment are indicated by hyphens. The amino acids conserved for the Rieske-type [2Fe-2S] cluster binding site and the mononuclear iron coordination site (28) are indicated by white type and shaded boxes, respectively. The DDBJ, EMBL, and GenBank nucleotide sequence accession numbers are indicated after the names of enzymes and the names of plasmids or bacterial strains (in parentheses).

FIG. 5

Terminal regions of ISs and their flanking sequences identified from strain CA10. The IRs and DRs are shown by white type and underlining, respectively. The complementary nucleotides between the left and right IRs are shown by asterisks. The direction of transposase genes is shown by the arrow.

FIG. 6

Comparison of 2,279-bp-long DNA regions (8,878 to 6,600 in AB047548), containing IS5car2 and the 5′ portion of the antA gene, with 2,115-bp-long DNA regions (25,306 to 27,420 in AB047548), containing IS5car1 and the 5′ portion of ORF9. ISs and ORFs are shown by the boxes. Shaded pentagons within the boxes represent the locations, lengths, and directions of translation of transposase genes. The hatched box represents the deleted region of the antA gene in the nucleotide sequence of ORF9. Dashed lines represent the neighboring region of transposed DNA.

FIG. 7

Southern blot analyses using the 332-bp PvuI-HindIII fragment (IS5car2) of pBCA711 (A), the 880-bp PstI-DraI fragment (IS5car3) of pBCA731 (B), and the 1,119-bp SmaI fragment (antA) of pBCA711 (C) as probes. Restriction endonucleases used for digestion of total DNA of strain CA10 are shown in the panels.

FIG. 8

Detection of CarD, CarF, and CarE produced by E. coli cells. Total cellular proteins of E. coli cells were analyzed by SDS-PAGE. Lanes: 1 and 8, molecular mass standards of 94, 67, 43, 30, 20.1, and 14.4 kDa (top to bottom, respectively); 2, JM109(pBCA616); 3, JM109[pBluescript II SK(−)]; 4, JM109(pUCA617); 5, JM109(pUC18); 6, JM109(pUCA618); 7, JM109(pUC19).

FIG. 9

Northern hybridization analysis using the antA gene as a probe. Total RNA was extracted from the CA10 cells grown on nutrient medium (lane 2) and CAR (lane 3) to late log phase. As the control, total RNA was similarly extracted from strain CA10 cells just after starvation culture (lane 1).

FIG. 10

Southern hybridization analysis of the chromosome and plasmid detected in strain CA10 cells using the 16S rRNA gene (EcoRI-PstI fragment of pTCA16S) (B), the car gene (EcoRI insert of pUCA1) (C), the cat gene (SalI fragment of pTE11) (D), and the tnpA2 gene (PvuI-HindIII fragment in IS5car2) (E) as probes. A photograph of the ethidium bromide-stained agarose gel is shown in panel A.

FIG. 11

Proposed model of the developmental process of the novel structure in the car gene cluster and its flanking region. Shaded boxes represent the car genes and several neighboring ORFs. Shaded boxes with triangles indicate the ISs. Striped boxes represent the ant genes or the transposed DNA region from the antA gene within ORF9. The arrows with solid lines indicate the transposition (recombination). The arrows with dashed lines indicate the duplication or homologous recombination.