Characterization of the basic replicon of Rhodococcus plasmid pSOX and development of a Rhodococcus-Escherichia coli shuttle vector.
The replication region of a 100-kb desulfurization plasmid (pSOX) from Rhodococcus sp. strain X309 was localized to a 4-kb KpnI fragment, and its sequence was determined. The amino acid sequence of one of the predicted open reading frames (ORFs) was related to the putative replication (Rep) protein sequences of the mycobacterial pLR7 family of plasmids. Three of the five predicted ORF products were identified by radiolabelling with the Escherichia coli T7 polymerase/promoter system. In E. coli, the Rep protein of pSOX was apparently synthesized in a shortened form, 21.3 kDa instead of the predicted 41.3 kDa, as a result of an internal initiation. This situation is reminescent of that for some bacterial Rep proteins. A shuttle plasmid was constructed with the pSOX origin, pBluescript II KS-, and the chloramphenicol resistance (Cmr) gene from pRF29. This new shuttle plasmid was used to demonstrate expression of the Bacillus subtilis sacB gene in a strain of Rhodococcus, rendering it sensitive to the presence of sucrose.
Denis-Larose
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Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, Canada H4P 2R2.
Bergeron
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H
Labbé
D
D
Greer
C W
CW
Hawari
J
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Grossman
M J
MJ
Sankey
B M
BM
Lau
P C
PC
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D006602:Hexosyltransferases / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012093:Replicon; D015183:Restriction Mapping; D012240:Rhodococcus / Q000235:genetics*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013395:Sucrose / Q000378:metabolism
D011994:Recombinant Proteins; D013395:Sucrose; D006602:Hexosyltransferases; C021513:levansucrase; C060630:endodeoxyribonuclease NaeI; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases; C029312:GGTACC-specific type II deoxyribonucleases