Characterization of the 450-kb linear plasmid in a polychlorinated biphenyl degrader, Rhodococcus sp. strain RHA1

Abstract

A strong polychlorinated biphenyl (PCB) degrader, Rhodococcus sp. strain RHA1, has diverse biphenyl/PCB degradative genes and harbors huge linear plasmids, including pRHL1 (1,100 kb), pRHL2 (450 kb), and pRHL3 (330 kb). The diverse degradative genes are distributed mainly on the pRHL1 and pRHL2 plasmids. In this study, the structural and functional characteristics of pRHL2 were determined. We constructed a physical map of pRHL2, and the degradative enzyme genes, including bphB2, etbD2, etbC, bphDEF, bphC2, and bphC4, were localized in three regions. Conjugal transfer of pRHL2 between RHA1 mutant derivatives was observed at a frequency of 7.5 x 10(-5) transconjugant per recipient. These results suggested that the linear plasmid is a possible determinant of propagation of the diverse degradative genes in rhodococci. The termini of pRHL2 were cloned and sequenced. The left and right termini of pRHL2 had 3-bp perfect terminal inverted repeats and were not as similar to each other (64% identity) as the known actinomycete linear replicons are. Southern hybridization analysis with pRHL2 terminal probes suggested that the right terminus of pRHL2 is similar to pRHL1 and pRHL3 termini. Retardation of both terminal fragments in the gel shift assay indicated that each terminus of pRHL2 is linked to a protein. We suggest that pRHL2 has invertron termini, as has been reported previously for Streptomyces linear replicons.

FIG. 1

PFGE of RHA1 linear plasmids performed with long (A) and short (B) pulse times. The positions of the chromosome and linear plasmids are indicated. (A) The pulse time was increased from 60 to 90 s during 23 h of electrophoresis. The voltage was adjusted to 6 V/cm. (B) The pulse time was increased from 20 to 30 s during 23 h of electrophoresis. The voltage was adjusted to 6 V/cm. Lane 1, S. cerevisiae chromosome marker, lane 2, total DNA of RHA1; lane 3, λ ladder marker.

FIG. 2

Physical map of pRHL2 and localization of degradative genes. The locations of degradative genes are indicated by bars below the map. The insert region of each pRHL2 subclone is also indicated below the map.

FIG. 3

Linear plasmids in transconjugants. Linear plasmids of a donor (RCA1), a recipient (RDO5), and transconjugants were separated by PFGE. The pulse time was increased from 60 to 90 s during 23 h of electrophoresis. The voltage was adjusted to 6 V/cm. Lane 1, S. cerevisiae chromosome marker; lane 2, RCA1; lane 3, RDO5; lanes 4 to 10, RDO5 transconjugants.

FIG. 4

Alignment of the terminal sequences of pRHL2 and pHG201. The dashes indicate gaps; the asterisks indicate identical nucleotide residues in pRHL2 and pHG201. The 3-bp perfect terminal inverted repeats are indicated by boldface type. The inverted repeats of pHG201 containing the control motif GCTXCGC which were identified by Kalkus et al. (11) are indicated by converging arrows.

FIG. 5

Southern hybridization of linear plasmids of RHA1 with the terminus probes of pRHL2. (A) Results of PFGE. The sizes of marker fragments and the positions of the chromosome and linear plasmids are indicated on the left and right, respectively. The pulse time was increased from 60 to 90 s during 23 h of electrophoresis. The voltage was adjusted to 6 V/cm. Lane 1, S. cerevisiae chromosome marker; lane 2, linear plasmids of RHA1. (B and C) Results of hybridization performed with the right (B) and left (C) end probes. The 1.1-kb EcoRI and 0.45-kb EcoRI-SalI fragments of pRHL2 were used as the right and left end probes, respectively. The lanes contained linear plasmids of RHA1. The bands of linear plasmids are indicated by arrowheads.

FIG. 6

(A and B) Normal PFGE (A) and SDS-PFGE (B) of linear plasmid DNAs obtained with and without proteinase K treatment. The pulse time was increased from 60 to 90 s during 23 h of electrophoresis. The voltage was adjusted to 6 V/cm. Lane 1, S. cerevisiae chromosome marker; lane 2, proteinase K-treated plasmid DNA; lane 3, non-proteinase K-treated plasmid DNA. The positions of the chromosome and plasmids are indicated on the left and right. (C to E) Results of PFGE (C) and hybridization (D and E) of total restriction fragments obtained with and without proteinase K treatment. Hybridization was performed with the right (D) and left (E) end probes. The 1.1-kb EcoRI and 0.45-kb EcoRI-SalI fragments of pRHL2 were used as the right and left end probes, respectively. The pulse time was increased from 3 to 12 s during 21 h of electrophoresis. The voltage was adjusted to 5.1 V/cm. The sizes of marker fragments in panel C are indicated on the left. The bands of interest in panels D and E are indicated by solid arrowheads. Minor bands in panel D which seemed to be derived from the termini of pRHL1 and pRHL3 are indicated by open arrowheads. Lane 1, non-proteinase K-treated AseI digests; lane 2, proteinase K-treated AseI digests; lane 3, λ ladder plus HindIII-digested λ DNA markers; lane 4, non-proteinase K-treated HpaI digests; lane 5, proteinase K-treated HpaI digests.