Identification of a novel composite transposable element, Tn5280, carrying chlorobenzene dioxygenase genes of Pseudomonas sp. strain P51

Abstract

Analysis of one of the regions of catabolic plasmid pP51 which encode chlorobenzene metabolism of Pseudomonas sp. strain P51 revealed that the tcbA and tcbB genes for chlorobenzene dioxygenase and dehydrogenase are located on a transposable element, Tn5280. Tn5280 showed the features of a composite bacterial transposon with iso-insertion elements (IS1066 and IS1067) at each end of the transposon oriented in an inverted position. When a 12-kb Hindlll fragment of pP51 containing Tn5280 was cloned in the suicide donor plasmid pSUP202, marked with a kanamycin resistance gene, and introduced into Pseudomonas putida KT2442, TnS280 was found to transpose into the genome at random and in single copy. The insertion elements IS1066 and IS1067 differed in a single base pair located in the inner inverted repeat and were found to be highly homologous to a class of repetitive elements of Bradyrhizobium japonicum and distantly related to IS630 of Shigella sonnei. The presence of the catabolic genes tcbA and tcbB on TnS280 suggests a mechanism by which gene clusters can be mobilized as gene cassettes and joined with others to form novel catabolic pathways. have been identified and characterized (49, 52) (Fig. 1). One region was found to contain a gene cluster, tcbCDEF, which encoded a modified ortho cleavage pathway (49, 52). The organization of the tcbCDEF gene cluster was found to be very similar to that of the tfdCDEF operon of Alcaligenes eutrophus JMP134 (8,14,34) and the clcABD operon of P. putida (11). The other region of pP51 contained the tcbAB gene cluster which encoded a chlorobenzene dioxygenase complex and a dehydrogenase, catalyzing the conversion of chlorobenzenes to chlorocatechols (52). This gene cluster exhibited size, organization, and function characteristics similar to those of other known bacterial dioxygenases, such as todCIC2BA from P. putida Fl (16,56), bphAB (12,23,30), or xylXYZ (20 respectively. E. coli S17-1 (41) was used as the donor strain for mobilization of pSUP202-derived plasmids. Pseudomonas putida KT2442 (hsdRl hsdM+) (10) was the recipient strain in mating experiments with E. coli S17-1 and was used to study transposition events. The strain is a rifampinresistant (Rif) plasmid-free derivative of strain P. putida mt-2. Plasmids pUC18 and pUC19 (55) were used as general

Characterization and expression of the plasmid-borne bedD gene from Pseudomonas putida ML2, which codes for a NAD+-dependent cis-benzene dihydrodiol dehydrogenase

“…There are several lines of evidence that lend support to the notion that the dehydrogenase reported here is the one utilized by P. putida ML2 in the catabolism of benzene. First, the contiguous organization of the dioxygenase and dehydrogenase genes is consistent with the molecular organization reported for other bacterial catabolic pathways (54,57,59). Such a gene arrangement would also support the possibility of a coupled dioxygenase-dehydrogenase complex in the intact organism (34,40).…”

Degradation of chloroaromatics by Pseudomonas putida GJ31: assembled route for chlorobenzene degradation encoded by clusters on plasmid pKW1 and the chromosome

“…The high mobility of upper pathway genes is further evidenced by the fact that the upper pathway genes on plasmid pP51 of Acidovorax sp. strain P51 (van der Meer et al, 1991) and in Ralstonia sp. strain PS12 (Beil et al, 1999;Müller et al, 2003) are carried on transposons, or are localized on genomic islands, as in Bordetella petrii (Gross et al, 2008).…”

IS870 requires a 5'-CTAG-3' target sequence to generate the stop codon for its large ORF1

“…The functional organization of IS870 and its closest relatives suggests an explanation for the unusual target site specificity. The large ORFs of the top strands of the IS870-like elements probably code for the transposase (7,37). The large ORFs of IS870, the A. rhizogenes element, and the R. fredii element lack a stop codon, but this codon is created by insertion into the CTAG target sequence (in the case of the A. rhizogenes element, the CTAG sequence at the left also creates a TAG stop codon for ORF7 on the other strand).…”