Complete nucleotide sequence of an exogenously isolated plasmid, pLB1, involved in gamma-hexachlorocyclohexane degradation

Abstract

The alpha-proteobacterial strain Sphingobium japonicum UT26 utilizes a highly chlorinated pesticide, gamma-hexachlorocyclohexane (gamma-HCH), as a sole source of carbon and energy, and haloalkane dehalogenase LinB catalyzes the second step of gamma-HCH degradation in UT26. Functional complementation of a linB mutant of UT26, UT26DB, was performed by the exogenous plasmid isolation technique using HCH-contaminated soil, leading to our successful identification of a plasmid, pLB1, carrying the linB gene. Complete sequencing analysis of pLB1, with a size of 65,998 bp, revealed that it carries (i) 50 totally annotated coding sequences, (ii) an IS6100 composite transposon containing two copies of linB, and (iii) potential genes for replication, maintenance, and conjugative transfer with low levels of similarity to other homologues. A minireplicon assay demonstrated that a 2-kb region containing the predicted repA gene and its upstream region of pLB1 functions as an autonomously replicating unit in UT26. Furthermore, pLB1 was conjugally transferred from UT26DB to other alpha-proteobacterial strains but not to any of the beta- or gamma-proteobacterial strains examined to date. These results suggest that this exogenously isolated novel plasmid contributes to the dissemination of at least some genes for gamma-HCH degradation in the natural environment. To the best of our knowledge, this is the first detailed report of a plasmid involved in gamma-HCH degradation.

FIG. 1.

Plasmids residing in UT26DB and its transconjugant UT26DBT1. Gel electrophoresis of plasmids (a) and Southern blot analysis with the linB gene as a probe (b) are shown. Lanes 1, UT26DB; lanes 2, UT26DBT1. The white arrowheads indicate endogenous plasmids pCHQ1 (185 kb), pUT1 (30 kb), and pUT2 (5 kb), respectively. The black arrowhead indicates pLB1.

FIG. 2.

Circular map of pLB1. CDSs or gene remnants outside the circle are transcribed in the clockwise direction and those inside in the counterclockwise direction. The putative functions of genes are shown by the following colors: red, replication and stable inheritance; orange, conjugative transfer; blue, transposase and resolvase; magenta, regulation; yellow, unknown; and green, others. Gene remnants are shown in gray. Large and small circles indicate the putative oriV and oriT regions, respectively. The region of the IS6100-linB cluster is shown by the light green arrow. The positions of tnp in the IS6100-linB cluster are almost equal to those of IS6100 (Table 2).

FIG. 3.

Structure of the putative oriV region of pLB1. The 9-bp direct repeats (5′-kCwAwCwsd-3′), which may serve as iterons, are shown by solid arrows, and one inverted repeat is indicated by dashed arrows. Putative DnaA-binding sequences are boxed, and parts of repA and orf21 are shaded. Putative ribosome-binding sites and start codons of orf21 and repA are shown in boldface.

FIG. 4.

Phylogenetic tree of putative Walker-type partition ATPases. Lengths of horizontal lines reflect relative evolutionary distances among the 33 Walker-type partition ATPases encoded by various plasmids or chromosomes. The GenBank accession numbers of respective proteins are shown in brackets. Groups of similar sequences are labeled. pLB1 is shown in boldface. The scale bar indicates 0.05 substitution per site.

FIG. 5.

Genetic organization of the putative transfer region of pLB1 and comparison with related systems. Genes encoding similar functions are displayed in the same color. Circles indicate the putative oriT regions of the respective plasmids. The numbers below the genes indicate the percent amino acid identity (>20%) to the corresponding gene product from pLB1. The lengths of TraO homologues are various, because only their C-terminal regions are conserved, which contain a putative functional domain for primase. The GenBank accession numbers of the respective nucleotide sequences are as follows: broad-host-range cryptic plasmid pIPO2, NC_003213; mercury resistance plasmid pSB102, NC_003122; A. tumefaciens strain C58 plasmid pTi, NC_003065; catabolic plasmid pWW0, NC_003350; and antibiotic resistance plasmid R388, BR000038.