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

Abstract

When Yersinia pseudotuberculosis is shifted from growth at 26 degrees C to growth at 37 degrees C, the synthesis of a plasmid-associated outer membrane protein, protein 1, is induced (Bölin et al., Infect. Immun. 37:506-512). 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 molecular weight as protein 1. Protein 1 was purified to homogeneity, and 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.