Multiple mechanisms contribute to lateral transfer of an organophosphate degradation (opd) island in Sphingobium fuliginis ATCC 27551

Abstract

The complete sequence of pPDL2 (37,317 bp), an indigenous plasmid of Sphingobium fuliginis ATCC 27551 that encodes genes for organophosphate degradation (opd), revealed the existence of a site-specific integrase (int) gene with an attachment site attP, typically seen in integrative mobilizable elements (IME). In agreement with this sequence information, site-specific recombination was observed between pPDL2 and an artificial plasmid having a temperature-sensitive replicon and a cloned attB site at the 3' end of the seryl tRNA gene of Sphingobium japonicum. The opd gene cluster on pPDL2 was found to be part of an active catabolic transposon with mobile elements y4qE and Tn3 at its flanking ends. Besides the previously reported opd cluster, this transposon contains genes coding for protocatechuate dioxygenase and for two transport proteins from the major facilitator family that are predicted to be involved in transport and metabolism of aromatic compounds. A pPDL2 derivative, pPDL2-K, was horizontally transferred into Escherichia coli and Acinetobacter strains, suggesting that the oriT identified in pPDL2 is functional. A well-defined replicative origin (oriV), repA was identified along with a plasmid addiction module relB/relE that would support stable maintenance of pPDL2 in Sphingobium fuliginis ATCC 27551. However, if pPDL2 is laterally transferred into hosts that do not support its replication, the opd cluster appears to integrate into the host chromosome, either through transposition or through site-specific integration. The data presented in this study help to explain the existence of identical opd genes among soil bacteria.

Keywords: catabolic transposons; genomic islands; integrative conjugative elements (ICE); organophosphates; phosphotriesterase (PTE).

Figure 1

Physical map of plasmid pPDL2. Outer and inner circles indicate proteins encoded by sense and anti-sense strands, respectively. Third circle indicates mobile elements and repeat sequences. Direct (DR) and Inverted (IR) repeats are shown with filled red and green triangles, respectively. Tn3-specific repeats appear with filled purple triangles in the fourth circle. The fifth circle shows GC content across the plasmid sequence. The sixth and seventh circles represent GC-skew in sense and anti-sense strands.

Figure 2

Replication module. Comparison of replicative origin (oriV) of plasmid pPDL2 (panel A) with oriV sequences of plasmids pUT1 (panel B) and pPS10 (panel C). Solid inverted arrows indicate inverted repeat sequences. The dnaA Box and iterons are shown with solid box and tandem arrows, respectively. The transcription orientation of repA is shown with a solid arrow. Filled triangles indicate repeats found in the A+T-rich region of pPS10.

Figure 3

Mobilization of pPDL2. Panel A represents secondary structure of predicted oriT sequence. The nick site that matches perfectly with the nick site of plasmid pLB1 of Sphingobium japonicum is shown in a dotted box. Affinity purification of RepB_C-6His is shown in panel B. Panel C indicates the mobility shift assay done using radiolabeled oriT fragment and purified RepB_C-6His. Lane 1 represents ³²P labeled oriT without RepB_C-6His. Lanes 2–5 represent labeled oriT incubated with increased concentrations [0.1 μg (2), 0.25 μg (3), 0.5 μg (4), and 1 μg (5)] of RepB_C-6His. Arrows indicate either free oriT or oriT-RepB_C-6His complex. OPH activity (i) and amplification of opd gene (ii) in exconjugants are shown in panel D.

Figure 4

Site-specific recombination. Integration of artificial plasmid pSDP8 at the predicted attP site of pPDL2. Panel A represents physical map of integration module. The underlined sequences indicate putative promoter elements. The CopG binding sites are shown with inverted arrows. The transcription orientation of integration modules I and II are shown with solid arrows. The synthetic attB site created using sequence of Sphingobium japonicum UT26S and the similarities between predicted pPDL2 attP and attB sequences are shown in (i) and (ii) of panel B, respectively. The physical map of pSDP8 and site of integration of pSDP8 in pPDL2 is shown in panel C. The restriction profile of the cointegrate (i) and the corresponding southern blot developed using either labeled pPDL2 (ii) or pSDP8 (iii) are shown in panel D. The increase in size of 4.7 kb PstI fragment due to integration of a 6.4 Kb plasmid pSD8 is shown with an arrow.

Figure 5

Transposition assay. Panel A indicates the physical map of the degradative module. The Tn3-specific terminal repeats found upstream and downstream of transposable element Tn3 and y4qE are shown with arrows. The reactions catalyzed by OPH, LigBA are shown. The transposon assay performed to assess transposition of the degradative module is shown in panel B. Panel C indicates affinity purification of LigBA and the corresponding Western blot using anti-His antibody. The dioxygenase activity of purified dioxygenase (LigBA) with various aromatic compounds is shown in panel D.