A 36-kilodalton Brucella abortus cell envelope protein is encoded by repeated sequences closely linked in the genomic DNA

Abstract

Recombinant bacteriophage expressing Brucella abortus antigens have been isolated from a lambda gt11 expression library by using antibody raised against a sodium dodecyl sulfate-polyacrylamide gel electrophoresis-purified cell envelope protein of 36 kilodaltons. Fusion products expressed by these recombinants vary in apparent molecular mass by sodium dodecyl sulfate-polyacrylamide gel electrophoresis but only slightly exceed the size of beta-galactosidase. Western blot (immunoblot) analysis of crude lysates derived from lambda gt11 lysogens indicates that the fusion products react specifically with the original antisera used for recombinant selection and selectively bind antibody directed against the 36-kilodalton cell envelope protein. Analysis of the DNA inserts from 11 independently selected recombinants reveals similar-size EcoRI fragments which range in size from 150 to 300 base pairs (bp), all of which cross-hybridize via Southern blot analysis. Three independently selected EcoRI inserts ranging in size from 200 to 270 bp have been subcloned into M13mp18 and sequenced; all three contain a common region of about 200 bp. Southern blot analysis of B. abortus genomic DNAs digested with EcoRI, PstI, or DdeI indicates the presence of two fragments which hybridize to these DNA probes while single BamHI and HindIII fragments hybridize. The absence of these sites from the internal DNA sequence of the cloned probes suggests the presence of more than one copy of these sequences within the B. abortus genome. The same DNA probes have been used to select genomic clones of approximately 20 kbp from a lambda 2001 library. The lambda 2001 recombinants contain single BamHI fragments and two PstI fragments which hybridize to these oligonucleotide probe constructed on the basis of the amino-terminal sequence of the mature gene product hybridizes to the same BamHI and PstI fragments as the lambda gt11-derived DNA probe. Although the relative positions of the oligonucleotide sequences and the lambda gt11 insert within the genes is not known, the two sequences flank a region which corresponds to at least 40% of the size of the predicted gene. Additional experimentation must be performed to determine whether these sequences represent either two complete structural genes encoding major cell envelope proteins or repetitive sequences within a single structural gene.