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Microbiology 150 (2004), 3075-3087; DOI  10.1099/mic.0.27217-0
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Microbiology 150 (2004), 3075-3087; DOI  10.1099/mic.0.27217-0
© 2004 Society for General Microbiology

A linear megaplasmid, p1CP, carrying the genes for chlorocatechol catabolism of Rhodococcus opacus 1CP

Christina König1, Dirk Eulberg2,{dagger}, Janosch Gröning1, Silvia Lakner2, Volker Seibert2,{ddagger}, Stefan R. Kaschabek1 and Michael Schlömann1

1 Interdisziplinäres Ökologisches Zentrum, Technische Universität Bergakademie Freiberg, Leipziger Str. 29, D-09599 Freiberg, Germany
2 Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany

Correspondence
Michael Schlömann
michael.schloemann{at}ioez.tu-freiberg.de

The Gram-positive actinobacterium Rhodococcus opacus 1CP is able to utilize several (chloro)aromatic compounds as sole carbon sources, and gene clusters for various catabolic enzymes and pathways have previously been identified. Pulsed-field gel electrophoresis indicates the occurrence of a 740 kb megaplasmid, designated p1CP. Linear topology and the presence of covalently bound proteins were shown by the unchanged electrophoretic mobility after S1 nuclease treatment and by the immobility of the native plasmid during non-denaturing agarose gel electrophoresis, respectively. Sequence comparisons of both termini revealed a perfect 13 bp terminal inverted repeat (TIR) as part of an imperfect 583/587 bp TIR, as well as two copies of the highly conserved centre (GCTXCGC) of a palindromic motif. An initial restriction analysis of p1CP was performed. By means of PCR and hybridization techniques, p1CP was screened for several genes encoding enzymes of (chloro)aromatic degradation. A single maleylacetate reductase gene macA, the clc gene cluster for 4-chloro-/3,5-dichlorocatechol degradation, and the clc2 gene cluster for 3-chlorocatechol degradation were found on p1CP whereas the cat and pca gene clusters for the catechol and the protocatechuate pathways, respectively, were not. Prolonged cultivation of the wild-type strain 1CP under non-selective conditions led to the isolation of the clc- and clc2-deficient mutants 1CP.01 and 1CP.02 harbouring the shortened plasmid variants p1CP.01 (500 kb) and p1CP.02 (400 kb).


Abbreviations: TIR, terminal inverted repeat

Dedicated to the memory of Olga Maltseva, who started the Rhodococcus work in our lab and persuaded us to follow up on it.

The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this article are AY569453 and AY569454.

{dagger}Present address: NOXXON Pharma AG, Max-Dohrn-Str. 8–10, D-10589 Berlin, Germany.

{ddagger}Present address: Europroteome AG, Neuendorfstraße 24a, D-16761 Hennigsdorf, Germany.




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