A linear megaplasmid, p1CP, carrying the genes for chlorocatechol catabolism of Rhodococcus opacus 1CP.
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).
König
Christina
C
Interdisziplinäres Ökologisches Zentrum, Technische Universität Bergakademie Freiberg, Leipziger Str. 29, D-09599 Freiberg, Germany.
Eulberg
Dirk
D
Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
Gröning
Janosch
J
Interdisziplinäres Ökologisches Zentrum, Technische Universität Bergakademie Freiberg, Leipziger Str. 29, D-09599 Freiberg, Germany.
Lakner
Silvia
S
Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
Seibert
Volker
V
Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
Kaschabek
Stefan R
SR
Interdisziplinäres Ökologisches Zentrum, Technische Universität Bergakademie Freiberg, Leipziger Str. 29, D-09599 Freiberg, Germany.
Schlömann
Michael
M
Interdisziplinäres Ökologisches Zentrum, Technische Universität Bergakademie Freiberg, Leipziger Str. 29, D-09599 Freiberg, Germany.
D002396:Catechols / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D005798:Genes, Bacterial; D062385:Hydroxybenzoates / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D009154:Mutation; D009693:Nucleic Acid Hybridization; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000235:genetics; D020161:Physical Chromosome Mapping; D010957:Plasmids; D012240:Rhodococcus / Q000235:genetics* / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D020079:Terminal Repeat Sequences
D002396:Catechols; D004269:DNA, Bacterial; D062385:Hydroxybenzoates; C031137:3-chlorocatechol; C063909:3,5-dichlorocatechol; C009091:protocatechuic acid; D044925:Oxidoreductases Acting on CH-CH Group Donors; C021530:maleylacetate reductase; D015719:Single-Strand Specific DNA and RNA Endonucleases; C038861:4-chlorocatechol