Isolation of a Brucella abortus Gene Encoding an Immunogenic 90kDa Cell Envelope Protein Using Transposon Mutagenesis

Abstract

A 16kb fragment of DNA containing the omp1 gene was isolated from Brucella abortus strain 19 and inserted into plasmid pBR322. The resultant plasmid, p1F8, was transformed into E. coli SE5000. Cells containing the plasmid were infected with bacteriophage λ carrying either transposon Tn5 or transposon TnphoA. Plasmid DNA from the mutants was isolated from the cells and cut with restriction endonucleases to determine the location of transposon insertion on p1F8, creating a physical map of the insertion site for each mutant. Mutants which had inserted in the general location of a previously mapped λgt11 recombinant were selected for further screening procedures. Cells transformed with TnphoA containing plasmids were screened for the presence of omp1/phoA fusion products, while both TnphoA and Tn5 mutants were screened for the expression of the omp1 gene product. One fusion product was detected out of a total of sixteen TnphoA insertions screened. Forty Tn5 and TnphoA mutants were screened for the expression of the OMPI protein: 25 showed continued expression, and 15 showed termination of expression. Data obtained in the screening procedures was correlated with the physical map of insertion sites to isolate the location of the omp1 gene between positions 6500 and 13000 on the DNA insert. Further investigation of the fusion product revealed the direction of transcription for the omp1 gene in the insert to be from right to left.