Differential activity of Rickettsia rickettsii opmA and ompB promoter regions in a heterologous reporter gene system.
The outer membrane of the Gram-negative obligate intracellular parasite Rickettsia rickettsii contains two large surface protein antigens with approximate molecular masses of 200 and 135 kDa termed rOmpA and rOmpB, respectively. rOmpB is the most abundant protein in the outer membrane, while rOmpA is a relatively minor constituent. Densitometry of intrinsically radiolabelled protein profiles from R. rickettsii-infected Vero cells indicated a molar ratio of approximately 1:9 between rOmpA and rOmpB. The putative promoter-5' untranslated regions (5' UTR) from their recently characterized genes (rompA and rompB) were placed in the promoter assay vector pKK232-8 to test whether these elements conserve aspects of differential expression in a heterologous host-reporter system. Primer extension analysis of RNA from Escherichia coli clones containing the constructs indicated that E. coli RNA polymerase faithfully utilizes rompA and rompB transcription start sites identified previously in R. rickettsii. The rompB insert directs 28-fold higher levels of chloramphenicol acetyl transferase activity than the rompA insert.
Policastro
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PF
Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, Montana 59840.
Hackstadt
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D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D012284:Rickettsia rickettsii / Q000235:genetics*; D014158:Transcription, Genetic
D001425:Bacterial Outer Membrane Proteins; D012333:RNA, Messenger; D015500:Chloramphenicol O-Acetyltransferase