Molecular cloning of the temperature-inducible outer membrane protein 1 of Yersinia pseudotuberculosis

Abstract

When Y. pseudotuberculosis is shifted from growth at 26.degree. C to growth at 37.degree. C, the synthesis of a plasmid-associated outer membrane protein, protein 1, is induced. The structural gene of this protein was found to be located on the virulence plasmid pIB1 of Y. pseudotuberculosis. One cosmid hybrid plasmid pBW8 was studied which carried a region of the virulence plasmid. This hybrid plasmid expressed in Escherichia coli K-12 a novel temperature-inducible outer membrane protein which is immunologically related to and has the same MW as protein 1. Protein 1 was purified to homogeneity; 14 amino acids of the N-terminal end were determined. From this sequence, the tentative corresponding DNA sequence was deduced; and a set of 11-nucleotide-long DNA probes was chemically synthesized. By using these probes in Southern blotting experiments, the genetic location of the N-terminal end of protein 1 was established. By introducing the transposon Tn5 into the virulence plasmid pIB1, mutants were obtained that did not express protein 1. One class of these mutants was still Ca2+ dependent and virulent, suggesting that protein 1 is not a major virulence determinant. Tn5-derived insertion mutants were also obtained which were Ca2+ independent. Such mutants were found to be avirulent. One Ca2+-independent mutant still expressed protein 1, indicating that the regulatory expression of protein 1 is not linked to Ca2+ dependence.