The Sphingomonas Plasmid pCAR3 Is Involved in Complete Mineralization of Carbazole

Abstract

We determined the complete 254,797-bp nucleotide sequence of the plasmid pCAR3, a carbazole-degradative plasmid from Sphingomonas sp. strain KA1. A region of about 65 kb involved in replication and conjugative transfer showed similarity to a region of plasmid pNL1 isolated from the aromatic-degrading Novosphingobium aromaticivorans strain F199. The presence of many insertion sequences, transposons, repeat sequences, and their remnants suggest plasticity of this plasmid in genetic structure. Although pCAR3 is thought to carry clustered genes for conjugative transfer, a filter-mating assay between KA1 and a pCAR3-cured strain (KA1W) was unsuccessful, indicating that pCAR3 might be deficient in conjugative transfer. Several degradative genes were found on pCAR3, including two kinds of carbazole-degradative gene clusters (car-I and car-II), and genes for electron transfer components of initial oxygenase for carbazole (fdxI, fdrI, and fdrII). Putative genes were identified for the degradation of anthranilate (and), catechol (cat), 2-hydroxypenta-2,4-dienoate (carDFE), dibenzofuran/fluorene (dbf/fln), protocatechuate (lig), and phthalate (oph). It appears that pCAR3 may carry clustered genes (car-I, car-II, fdxI, fdrI, fdrII, and, and cat) for the degradation of carbazole into tricarboxylic acid cycle intermediates; KA1W completely lost the ability to grow on carbazole, and the carbazole-degradative genes listed above were all expressed when KA1 was grown on carbazole. Reverse transcription-PCR analysis also revealed that the transcription of car-I, car-II, and cat genes was induced by carbazole or its metabolic intermediate. Southern hybridization analyses with probes prepared from car-I, car-II, repA, parA, traI, and traD genes indicated that several Sphingomonas carbazole degraders have DNA regions similar to parts of pCAR3.

Characterization and genetic analyses of a carbazole-degrading gram-positive marine isolate, Janibacter sp. strain OC11

“…Strain KA1 is known to lose the plasmid pCAR3 containing car genes and its CAR-degrading ability when cultured in nutrient broth. 30,31) These characteristics are similar to our findings for strain OC11. Additionally, in the meta-cleavage enzyme test, transformed E. coli clones containing carBaBb turned the medium yellow, confirming the presence of meta-cleavage enzyme activity.…”

“…In addition, strains CA10, KA1, and IC177 have transposons, and strain CA10 also has the plasmid pCAR1. 7,31,32) These facts indicate that OC11 most likely obtained car OC11 through horizontal transmission using mobile DNA, such as transposons and plasmids. This speculation is also supported by the result that the car genes from strain OC11 showed high homology to those of strain IC177.…”

“…KA1, which are gramnegative, and Nocardioides aromaticivorans IC177, which is gram-positive. 1,[4][5][6][7] The car gene clusters of these strains comprise carAa, carAc, and carAd (however, carAd of strain KA1 has not been identified to date), which encode a CAR 1,9a-dioxygenase (CARDO); carBa and carBb, the small and large subunits of the meta-cleavage enzyme; and carC, the meta-cleavage compound hydrolase. 8,9) The car genes of strains CA10 and KA1 are organized as carAaBaBbCAcAd and carAaBaBbCAc, respectively, while those of strain IC177 are carAaCBaBbAcAd (Fig.…”

Obligate oil-degrading marine bacteria

“…3(B), the genetic organization of the phn gene cluster of the gamma-proteobacterial Cycloclasticus is similar to that found in several alphaproteobacteria. The phn gene cassette of Novosphingobium aromaticivorans F199 is located on a large plasmid, pNL1 [54]. Interestingly, the pdxA gene, which specifies an enzyme involved in the biosynthesis of coenzyme PLP, is clustered and co-oriented with phnA1b, seemingly a characteristic feature of phn-like cassettes (Figure 3(B)).…”

Structural and Molecular Genetic Analyses of the Bacterial Carbazole Degradation System

“…J3, 19) Novosphingobium sp. KA1, 20,21) and Nocardioides aromaticivorans IC177. 22,23) CARDO is a three-component dioxygenase system consisting of a terminal oxygenase and electron transport proteins (Fig.…”