Characterization of the 101-Kilobase-Pair Megaplasmid pKB1, Isolated from the Rubber-Degrading Bacterium Gordonia westfalica Kb1

Abstract

The complete sequence of the circular 101,016-bp megaplasmid pKB1 from the cis-1,4-polyisoprene-degrading bacterium Gordonia westfalica Kb1, which represents the first described extrachromosomal DNA of a member of this genus, was determined. Plasmid pKB1 harbors 105 open reading frames. The predicted products of 46 of these are significantly related to proteins of known function. Plasmid pKB1 is organized into three functional regions that are flanked by insertion sequence (IS) elements: (i) a replication and putative partitioning region, (ii) a putative metabolic region, and (iii) a large putative conjugative transfer region, which is interrupted by an additional IS element. Southern hybridization experiments revealed the presence of another copy of this conjugational transfer region on the bacterial chromosome. The origin of replication (oriV) of pKB1 was identified and used for construction of Escherichia coli-Gordonia shuttle vectors, which was also suitable for several other Gordonia species and related genera. The metabolic region included the heavy-metal resistance gene cadA, encoding a P-type ATPase. Expression of cadA in E. coli mediated resistance to cadmium, but not to zinc, and decreased the cellular content of cadmium in this host. When G. westfalica strain Kb1 was cured of plasmid pKB1, the resulting derivative strains exhibited slightly decreased cadmium resistance. Furthermore, they had lost the ability to use isoprene rubber as a sole source of carbon and energy, suggesting that genes essential for rubber degradation are encoded by pKB1.The genus Gordonia was proposed by Tsukamura for coryneform bacteria isolated from sputa of patients with pulmonary disease or from soil (65-67). Gordonia belongs to the so-called CMN group (Corynebacterium, Mycobacterium, and Nocardia) of actinomycetes, which synthesize mycolic acids (13, 59). Gordonia strains also play an important role in bioremediation and biodegradation of pollutants and have attracted much interest in recent years due to their unusual and diverse metabolic capabilities (23,29,30,70). Three strains of Gordonia polyisoprenivorans (2, 37) and the novel species G. westfalica Kb1 (39) were described as bacteria able to degrade natural rubber and synthetic cis-1,4-polyisoprene, which allows species of this genus to serve as model organisms for the investigation of the hitherto unknown biochemical and molecular mechanisms of rubber biodegradation (36). So far, no native plasmids of the genus Gordonia have been detected. Since factors encoded by linear or circular plasmids are often involved in degradation of complex xenobiotics (62), rubberdegrading bacterial strains were screened for the occurrence of extrachromosomal DNA. This publication gives the first example and the complete DNA sequence of a native Gordonia plasmid, which also provides the basis for the development of shuttle vectors that may serve as a novel genetic tool for the study of Gordonia and related genera. MATERIALS AND METHODSBacterial strains, plasmids, and cultivation conditio...

Involvement of Two Latex-Clearing Proteins during Rubber Degradation and Insights into the Subsequent Degradation Pathway Revealed by the Genome Sequence of Gordonia polyisoprenivorans Strain VH2

“…Except for the second lcp and a gene encoding an enoyl-CoA hydratase (see above), the plasmid does not exhibit genes that can be associated with the proposed rubber degradation pathway. p174 is considerably different from the plasmid pKB1, which was shown to be essential for rubber degradation by G. westfalica (20). However, the plasmid also harbors genes that might play a role in genetic information storage and processing (including many transposases) as well as in metabolism (such as uptake and utilization of amino acids and carbon metabolism).…”

TraA is required for megaplasmid conjugation in Rhodococcus erythropolis AN12

“…The TraA (Genbank Accession No. CAE09129) from Gordonia westfalica is encoded by a 101 kb megaplasmid called pKB1 (Broker et al, 2004), and is 31% identical to pREA400 TraA over 1079 residues. It is unclear whether the traA gene product (Genbank Accession No.…”

Evolution of theRhodococcus equi vapPathogenicity Island Seen through Comparison of Host-AssociatedvapAandvapBVirulence Plasmids

“…The conserved genes include a pVAPA/B_0310/pREC1_0059 (putative phage excisionase) homolog located in a similar position in the replication module, providing further evidence of a common origin for pKB1 and the rhodococcal plasmids. The pKB1-specific VR encodes products putatively involved in heavy metal ion detoxification and redox processes, together with many hypothetical or membrane proteins (8). As in the rhodococcal plasmids, the VR is also flanked by mobility genes, but their putative products (a transposase and a bacteriophagerelated protein) are different from the resA-and invA-like gene FIG.…”

“…4. ACT analysis of the rhodococcal pVAP and pREC1 plasmids and Gordonia westfalica pKB1 plasmid (8). See the legends to Fig.…”